Elongated and multiple spacers in activatible prodrugs

ABSTRACT

This invention is directed to prodrugs that can be activated at the preferred site of action in order to selectively deliver the corresponding therapeutic parent drugs to target cells or to the target site. This invention will therefore primarily but not exclusively relate to tumor cells as target cells. More specifically the prodrugs are compounds of the formula V—(W) k —(X) l —A—Z, wherein: V is a specifier; (W) k —(X) l —A is an elongated self-elimination spacer system; W and X are each a 1,(4+2n) electronic cascade spacer, being the same or different; A is either a spacer group of formula (Y) m  wherein: Y is a 1,(4+2n) electronic cascade spacer, or a group of formula U being a cyclization elimination spacer; Z is a therapeutic drug; k, l and m are integers from 0 (included) to 5 (included); n is an integer of 0 (included) to 10 (included), with the provisos that: —when A is (Y) m : k+l+m≧1, and if k+l+m=1; —when A is U: k+l≧1.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the priority of EP-A-01201095.5, filed 23 Mar.2001, publication No. EP 1 243 276, the disclosure thereof beingincorporated by reference.

FIELD OF THE INVENTION

This invention is directed to prodrugs that can be activated at thepreferred site of action in order to selectively deliver thecorresponding therapeutic or diagnostic parent moiety to target cells orto the target site. This invention will therefore primarily but notexclusively relate to tumor cells as target cells.

BACKGROUND OF THE INVENTION

Lack of selectivity of chemotherapeutic agents is a major problem incancer treatment. Because highly toxic compounds are used in cancerchemotherapy, it is typically associated with severe side effects. Drugconcentrations that would completely eradicate the tumor cannot bereached because of dose-limiting side effects such as gastrointestinaltract and bone marrow toxicity. In addition, tumors can developresistance against anticancer agents after prolonged treatment. In modemdrug development, targeting of cytotoxic drugs to the tumor site can beconsidered one of the primary goals.

A promising approach to obtain selectivity for tumor cells or tumortissue is to exploit the existence of tumor-associated enzymes. Arelatively high level of tumor-specific enzyme can convert apharmacologically inactive prodrug to the corresponding active parentdrug in the vicinity of the tumor. Via this concept a high concentrationof toxic anticancer agent can be generated at the tumor site. All tumorcells may be killed if the dose is sufficiently high, which may decreasedevelopment of drug resistant tumor cells.

There exist several enzymes that are present at elevated levels incertain tumor tissues. One example is the enzyme β-glucuronidase, whichis liberated from certain necrotic tumor areas. Furthermore, severalproteolytic enzymes have been shown to be associated with tumor invasionand metastasis. Several proteases, like for example the cathepsins andproteases from the urokinase-type plasminogen activator (u-PA) systemare all involved in tumor metastasis. The serine protease plasmin playsa key role in tumor invasion and metastasis. The proteolytically activeform of plasmin is formed from its inactive pro-enzyme form plasminogenby u-PA. The tumor-associated presence of plasmin can be exploited fortargeting of plasmin-cleavable prodrugs.

In this invention a new technology is disclosed that can be applied toprepare improved prodrugs or conjugates for targeting drugs todisease-related or organ-specific tissue or cells, for exampletumor-specific prodrugs. This technology can furthermore findapplication in (non-specific) controlled release of compounds, with theaim of facilitating release of the parent moiety. The present inventionis deemed to be applicable to all drugs that need to be delivered at aspecific target site where a specific disease-related biomolecule canconvert the prodrug into the drug or induce conversion of the prodruginto the drug.

DESCRIPTION OF THE INVENTION

The technology of this invention relates to novel linker systems to beinserted between a specifier (=part of prodrug to be cleaved by theenzyme) and parent drug. A great number of anticancer prodrugs that havebeen developed in the past contain a self-eliminating connector orlinker, also called self-elimination spacer. This spacer is incorporatedbetween the specifier and the drug in order to facilitate enzymaticcleavage and so enhance the kinetics of drug release (as shown in FIG.1). The specifier (which for example can be an oligopeptide substratefor a protease or for example a β-glucuronide substrate forβ-glucuronidase) must be site-specifically removed, followed by aspontaneous spacer elimination to release the cytotoxic parent drug. Inthis invention, greatly improved linker systems are disclosed. These areapplicable in prodrugs, for example anticancer prodrugs, andsignificantly enhance enzymatic activation rates.

More specifically, the invention relates to compounds of the formula:V—(W)_(k)—(X)_(l)—A—Zwherein:

-   V is an enzymatically removable specifier,-   (W)_(k)—(X)_(l)—A is an elongated self-eliminating spacer system,-   W and X are each a 1,(4+2n) electronic cascade spacer, being the    same or different,-   A is either a spacer group of formula (Y)_(m), wherein Y is a    1,(4+2n) electronic cascade spacer, or a group of formula U, being a    cyclisation elimination spacer,-   Z is a therapeutic or diagnostic moiety,-   k, l and m are independently an integer of 0 (included) to 5    (included),-   n is an integer of 0 (included) to 10 (included), with the provisos    that:    -   when A is (Y)_(m): then k+l+m≧1, and if k+l+m=1, then n>1;    -   when A is U: then k+l≧1.

These novel elongated linker systems show improved enzymatic activationcharacteristics, which is demonstrated in the following Examples.

An activatible prodrug according to this invention comprises a specifierV, which is meant to consist of a group that can be site specificallyremoved and that is covalently attached to a therapeutic or diagnosticmoiety Z via the novel elongated self-eliminating connector system(W)_(k)—(X)_(l)—A of the invention (FIG. 2). These self-eliminatingconnector systems possess increased lengths, which places the parentmoiety Z at an increased distance from the specifier.

It is observed that spacers which self-eliminate through a1,(4+2n)-elimination (n=0,1,2,3,4,5 . . . 10) (for example1,6-elimination, 1,8-elimination, or 1,10-elimination) are from nowcalled ‘electronic cascade’ spacers.

According to a preferred embodiment of the invention are the electroniccascade spacers W, X, and Y independently selected from compounds havingthe formula:

Q=—R⁵C═CR⁶—, S, O, NR⁵, —R⁵C═N—, or —N═CR⁵—P=NR⁷, O, Swherein

-   a, b, and c are independently an integer of 0 (included) to 5    (included);-   I, F and G are independently selected from compounds having the    formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ independently representH, C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy(OH), amino (NH₂), mono-substituted amino (NR_(x)H), di-substitutedamino (NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me,CONHMe, cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl,morpholino, thiol (SH), thioether (SR_(x)), tetrazole, carboxy (COOH),carboxylate (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)),sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)),sulphinyl (S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate(OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² are independentlyselected from a C₁₋₆ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀aryl group, two or more of the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, or R⁹ optionally being connected to one another to form one or morealiphatic or aromatic cyclic structures.

It is further observed that the principle of 1,6-elimination, as suchdeveloped in 1981, can be considered one of the most versatileself-elimination principles that can be used in prodrug design.According to this principle, spacer elimination proceeds via themechanism depicted in FIG. 3. This particular elimination process hasproven to be very successful when applied in the prodrug concept.Spacers that self-eliminate through an electronic cascade sequence asindicated in FIG. 3 generally show much faster half-lives of eliminationthan do spacers that eliminate via a cyclisation reaction. This is asignificant difference between cyclization spacers and electroniccascade spacers.

In the following Examples, para-aminobenzyloxycarbonyl (PABC) andrelated electronic cascade spacer systems are used because theyeliminate more rapidly upon unmasking of the amine, when compared tohydroxybenzyl-based electronic cascade spacers, which needelectron-withdrawing substituents on the phenyl part of the spacer inorder to let spacer elimination take place. Drug release will not takeplace when the spacer is an un-substituted hydroxybenzyl electroniccascade spacer.

Most efforts were, in the past, directed to the synthesis of electroniccascade spacers containing electron-withdrawing substituent(s). It washypothesized that the withdrawal of electrons from the site whereenzymatic activation occurs would enhance the rate of enzymaticactivation. However, the activation rate of prodrugs containing anelectron-withdrawing group on the spacer is usually not significantlydifferent from that of un-substituted electronic cascade spacercontaining prodrugs: A chloro-substituent on an aminobenzyl spacer forexample only marginally enhances the rate of enzymatic prodrugactivation by plasmin. In the case of aminobenzyl spacer-containinganthracycline prodrugs for activation by β-glucuronidase, chloro- orbromo-substituents showed only a marginal effect. It must further beconsidered that, although electron-withdrawing substituents onaminobenzyl spacers may increase enzymatic activation rates, spacerelimination rates will decrease as a consequence of substituents withsuch electronic properties. In the case of generation of hydroxylaminobenzyl electronic cascade spacers it appeared that indeedelectron-donating substituents on the benzyl ring acceleratedfragmentation. This effect can probably be ascribed to stabilization ofthe developing positive charge on the benzylic carbon by thesesubstituents. In some cases, when one or more of the spacer substituentsare too electron-withdrawing, spacer elimination will not occur at all.An aminobenzyl spacer containing a nitro substituent at the metaposition with respect to the specifier did not self-eliminate to releasethe free drug. It was also found that a hydroxylamino benzyl spacer witha nitro substituent at the meta position with respect to the specifiershowed the slowest spacer elimination rate of such substitutedhydroxylamino benzyl spacers. It appears that electron-withdrawingproperties of spacer substituents have only marginal impact on enzymaticactivation rates, whereas spacer elimination is greatly dependent onelectronic properties of spacer substituents and occurs only in arelatively narrow characteristic electronic profile depending on thetype of cascade spacer that is used.

In several previously reported prodrugs, containing one electroniccascade spacer, differences in enzymatic activation rates can still beobserved when different parent drugs are connected with the samepromoiety or when a parent drug is connected to the same promoiety via adifferent site of the drug. For example, β-glucuronidase cleaves theglucuronide from a β-glucuronide-cyclisation spacer promoiety muchslower when paclitaxel is the parent drug in comparison with the prodrugcontaining doxorubicin as the parent drug. In another example, adipeptide derivative of paclitaxel, linked via an aminobenzyl spacer wasmore readily cleaved by cathepsin B when paclitaxel was linked via its7-position than via its 2′-position. In addition, half-lives ofcathepsin B cleavage of electronic cascade spacer containing prodrugs ofdoxorubicin or mitomycin C were much shorter than the half-life of thecorresponding prodrugs with paclitaxel as the parent drug. Finally,plasmin cleaves the tripeptide from an electronic cascade spacercontaining doxorubicin prodrug much more readily than the tripeptidefrom the corresponding paclitaxel prodrug. Thus, in several prodrugsystems the parent drug still exerts a significant effect on the rate ofenzymatic activation, even though the mentioned prodrugs all containedone electronic cascade spacer.

The invention obviates the above-mentioned drawbacks by reduction of theinfluence of substituents of the spacer group on prodrug activationand/or spacer elimination and of the parent drug on the rate ofenzymatic activation of the prodrug due to the presence of elongatedspacer systems.

The invention is in a second aspect related to compounds of theabove-mentioned formula wherein group U is a cyclisation spacer, fromnow called ‘ω-amino aminocarbonyl’ cyclisation spacer, and Z is amolecule having a hydroxyl group.

More preferably, the ω-amino aminocarbonyl cyclisation eliminationspacer U of the invention is a compound having the formula:

wherein:

-   a is an integer of 0 or 1; and-   b is an integer of 0 or 1; and-   c is an integer of 0 or 1; provided that-   a+b+c=2 or 3;-   and wherein R¹ and/or R² independently represent H, C₁₋₆ alkyl, said    alkyl being optionally substituted with one or more of the following    groups: hydroxy (OH), ether (OR_(x)), amino (NH₂), mono-substituted    amino (NR_(x)H), disubstituted amino (NR_(x) ¹R_(x) ²), nitro (NO₂),    halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe, cyclic C₁₋₅ alkylamino,    imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino, thiol (SH), thioether    (SR_(x)), tetrazole, carboxy (COOH), carboxylate (COOR_(x)),    sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)), sulphonyl    (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)),    sulphinyl (S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate    (OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² are selected    from a C₁₋₆ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀ aryl    group; and-   R³, R⁴, R⁵, R⁶, R⁷, and R⁸ independently represent H, C₁₋₄ alkyl,    C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy (OH), amino    (NH₂), mono-substituted amino (NR_(x)H), disubstituted amino (NR_(x)    ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe,    cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl,    morpholino, thiol (SH), thioether (SR_(x)), tetrazole, carboxy    (COOH), carboxylate (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate    (S(═O)₂OR_(x)), sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH),    sulphinate (S(═O)OR_(x)), sulphinyl (S(═O)R_(x)), phosphonooxy    (OP(═O)(OH)₂), and phosphate (OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹    and R_(x) ² are selected from a C₁₋₆ alkyl group, a C₃₋₂₀    heterocyclyl group or a C₅₋₂₀ aryl group, two or more of the    substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ optionally being    connected to one another to form one or more aliphatic or aromatic    cyclic structures.

In a third aspect, the invention relates to compounds of theabove-mentioned formula wherein spacer group A is an electronic cascadespacer having the formula:

Q=—R⁵C═CR⁶—, S, O, NR⁵, —R⁵C═N—, or —N═CR⁵—P=NR⁷, O, Swherein

-   a, b, and c are independently an integer of 0 to 5;-   I, F and G are independently selected from compounds having the    formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ independently representH, C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy(OH), amino (NH₂), mono-substituted amino (NR_(x)H), disubstituted amino(NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe,cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino,thiol (SH), thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate(COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)), sulphonyl(S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)), sulphinyl(S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate(OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² are independentlyselected from a C₁₋₄ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀aryl group, two or more of the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, or R⁹ optionally being connected to one another to form one or morealiphatic or aromatic cyclic structures.

In one embodiment the elongated spacer system (W)_(k)—(X)_(l)—A is amolecule that self-eliminates via a 1,(4+2n)-elimination (n=2,3,4,5 . .. 10), for example a 1,8-elimination (FIG. 4). The length of this novelspacer system can be extended, for example to a 1,10-elimination spacersystem, in which two or more double or triple bonds instead of one areconjugated with the aromatic part of the spacer (FIG. 5).

In another embodiment, the spacer system of the invention consists oftwo or more electronic cascade spacers that are connected to oneanother. Release of the leaving group (the drug) occurs after two ormore subsequent spacer eliminations.

In again another embodiment, prodrugs of hydroxyl functionalitycontaining drugs (such as for example paclitaxel) are claimed thatcontain both one or more electronic cascade spacers and a cyclisationspacer.

In a preferred embodiment the spacer that is directly connected to thepaclitaxel molecule is an ω-amino aminocarbonyl cyclisation spacer thatis linked to the 2′-position of paclitaxel via a carbamate linkage. Aconvenient synthetic route to this class of paclitaxel derivatives isdisclosed.

In another embodiment the elongated electronic cascade spacer system iscoupled to the phenolic hydroxyl group of the drug moiety via an etherlinkage. When the leaving group (i.e., the drug) is an phenolic hydroxylgroup, a para-aminobenzylether has been reported to self-eliminate.

The elongated spacer systems provide for improved enzymatic activationcharacteristics.

The self-eliminating connector systems in this invention possessincreased lengths with respect to an electronic cascade spacer systemavailable at present. It is observed that one end of the linker systemmust be able to react with the specifier, for example the tripeptidethat is a substrate for plasmin. Typically, this end of the spacersystem is an amino group or a hydroxyl group, but it can also be anotherfunctionality. The functionality at the other end of the linker systemmust be able to react with the drug. Typically, this end of the spacersystem is a hydroxyl group, but it can also be another functionality. Inone embodiment this functionality reacts with an amino group of the drugto form a carbamate linkage between linker and drug. In anotherembodiment, this functionality reacts with a hydroxyl group of the drugto form a carbonate linkage between linker and drug. In again anotherembodiment, this functionality reacts with a sulfhydryl group of thedrug to form a thiocarbonate linkage between linker and drug. In againanother embodiment this functionality reacts with a carboxylic acidgroup of the drug to form an ester linkage between linker and drug.

Typically, the spacer system isp-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminocinnamyloxycarbonyl,p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminobenzyloxycarbonyl-p-aminocinnamyloxycarbonyl,p-aminocinnamyloxycarbonyl-p-amino-cinnamyloxycarbonyl,p-aminophenylpentadienyloxycarbonyl,p-aminophenylpenta-dienyloxycarbonyl-p-aminocinnamyloxycarbonyl,p-aminophenylpentadienyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyloxycarbonyl,p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminobenzyloxycarbonyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminobenzyloxycarbonyl-p-aminobenzyl,p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyl,p-aminocinnamyl, p-aminocinnamyloxycarbonyl-p-aminobenzyl,p-aminobenzyloxycarbonyl-p-aminocinnamyl,p-aminocinnamyloxycarbonyl-p-aminocinnamyl, p-aminophenylpentadienyl,p-aminophenylpentadienyloxycarbonyl-p-aminocinnamyl,p-aminophenylpentadienyloxycarbonyl-p-aminobenzyl, orp-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyl.

In the compounds of formula V—(W)_(k)—(X)_(l)—A—Z, the specifier Vtypically contains a substrate molecule that is specifically cleaved byan enzyme present in the vicinity of the target cells, for example tumorcells. More preferably, the specifier V contains a substrate that isspecifically cleaved by an enzyme present at elevated levels in thevicinity of the target cells as compared to other parts of the body, andmost preferably the enzyme is present only in the vicinity of the targetcells.

The specifier V may also contain a moiety that targets the compounds offormula V—(W)_(k)—(X)_(l)—A—Z to the target site by selective complexingwith a receptor or other receptive moiety associated with a given targetcell population or by causing accumulation of compoundsV—(W)_(k)—(X)_(l)—A—Z in the vicinity of the target cells by anothermechanism. This targeting moiety may, for example, be bombesin,transferrin, gastrin, gastrin-releasing peptide, a molecule thatspecifically binds α_(v)β₃ and/or α_(v)β₅-integrin receptors, such asRGD-containing peptides, platelet-derived growth factor, IL-2, IL-6, atumor growth factor, vaccinia growth factor, insulin and insulin-likegrowth factors I en II, an antigen-recognizing immunoglobulin or anantigen-recognizing fragment thereof, or a carbohydrate. Preferably,that antigen recognized by the immunoglobulin (or fragment thereof) isspecific for the target cells, e.g. a tumor-specific antigen. Thespecifier V may also contain a polymer, which causes accumulation ofcompounds V—(W)_(k)—(X)_(l)—A—Z in the vicinity of the target cells,e.g. tumor cells, because of the Enhanced Permeability and Retention(EPR) effect.

In one embodiment, the specifier is a di-, tri-, or oligopeptide whichconsists of an amino acid sequence specifically recognized by aprotease, for example plasmin, a cathepsin, cathepsin B,prostate-specific antigen (PSA), urokinase-type plasminogen activator(u-PA), or a member of the family of matrix metalloproteinases, presentin the vicinity of the target cells, for example tumor cells, or, inanother embodiment, a β-glucuronide that is specifically recognized byβ-glucuronidase present in the vicinity of tumor cells. In again anotherembodiment the specifier is a nitro-aromatic moiety that can be reducedunder hypoxic conditions or by nitroreductases. After removal of thenitro-aromatic specifier, elimination of the spacer systems described inthis invention leads to drug release. It can be understood that anyspecifier that is specifically cleaved following recognition by adisease-specific and/or organ-specific enzyme and/or receptor can beincorporated into prodrugs that contain the linker systems claimed inthis invention.

The moiety Z is a therapeutic or diagnostic moiety. Z can for instancebe an anticancer drug, an antibiotic, an anti-inflammatory agent, or ananti-viral agent. Typically, the moiety Z is an anticancer drug.Preferably the anticancer drug is the amino containing daunorubicin,doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, an anthracycline,mitomycin C, mitomycin A, 9-amino campiothecin, aminopterin,actinomycin, bleomycin, N⁸-acetyl spermidine,1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine, tallysomycin, orderivatives thereof. The anticancer drug can also be the hydroxylcontaining etoposide, camptothecin, irinotecan, topotecan, 9-aminocamptothecin, paclitaxel, docetaxel, esperamycin,1,8-dihydroxybicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one, anguidine,doxorubicin, morpholinedoxorubicin, N-(5,5-diacetoxypentyl) doxorubicin,vincristine, vinblastine, or derivatives thereof. The anticancer drugcan also be the sulfhydryl containing esperamicin, 6-mercaptopurine, orderivatives thereof. The drug can also be the carboxyl containingmethotrexate, camptothecin (ring-opened form of the lactone), butyricacid, retinoic acid, or derivatives thereof.

To show the principle of elimination of elongated spacer systems,tumor-specific prodrugs that are selectively hydrolyzed by thetumor-associated protease plasmin were synthesized. The synthesizedprodrugs consist of a tripeptide specifier that is coupled to the drugvia an elongated self-eliminating spacer. The tripeptide specifiercontains an amino acid sequence that is specifically recognized by thetumor-associated enzyme plasmin. The synthesis of these derivatives isdisclosed.

There is an increasing body of literature that links production ofcertain proteases to tumor malignancy. Mostly, proteolytic activity isrequired for tumor cells when they become invasive and form metastases.A primary tumor is encapsulated in an extracellular matrix, whichconsists of proteins. In order to form metastases, the primary tumormust break through this matrix. For this reason, enhanced expression ofproteolytic enzymes by invading and metastasizing tumors is generated.Recent studies indicate that proteases are involved also in earlierstages of tumor progression, at both primary and metastatic sites. Anumber of proteases, like cathepsins, the u-PA system, and the matrixmetalloproteinases, take part in the proteolytic cascade.

The u-PA system has received broad attention in the literature,especially in the last decade. Several invasive and metastasizing humantumors express a significantly higher plasminogen activator activity incomparison with normal tissue. An increased activity and expression ofu-PA is found in several tumor cell lines and human solid tumors, likelung tumors, prostate cancers, breast cancers, ovarian carcinomas andseveral other cancer types. u-PA is an important enzyme in proteolyticreactions that are required for the spreading and invasiveness of cells,both in cancer and in tissue remodeling processes. u-PA interacts with aspecific high-affinity receptor on the cell surface. Receptor-bound u-PAis catalytically active on the surface of the cell without requiringinternalization. It interacts with plasminogen to produce plasmin thatis still bound to the cell surface. The high u-PA level via this pathwayleads to elevated levels of plasmin. There exists substantial evidencethat the protease plasmin itself plays a key role in tumor invasion andmetastasis. Plasmin itself catalyses the breakdown of extracellularmatrix proteins. Thus, the plasminogen activator system is intimatelyassociated with tumor metastasis. Even the process of angiogenesis,nowadays considered as an important target mechanism for the developmentof new therapeutic strategies, is a urokinase dependent process. Theplasminogen activation system may be involved in cell adhesion processesby regulating integrin functions. Vascular endothelial growth factor(VEGF), an angiogenic molecule, is suggested to interact with u-PA intumor progression. u-PA catalyzed plasmin generation proved to be animportant determinant of tumor metastasis in many experiments withanimal model systems.

For the reasons outlined above, plasmin can be a very promising enzymefor the targeting of peptide prodrugs of anticancer agents. Activeplasmin is localized in tumor tissue because it is formed from itsinactive pro-enzyme form plasminogen by u-PA, produced by cancer and/orstroma cells. In the blood circulation active plasmin is rapidlyinhibited by inhibitors that block the active site, such asα₂-antiplasmin. Cell-bound plasmin as present in tumor tissue is notinhibited. In addition, plasmin is suitable as a target enzyme forprodrugs, because it is generated at the end of the proteolytic cascade.One molecule of u-PA can generate more than one molecule of plasmin.

The amino acid sequence of the tripeptide to be a plasmin substrate mustbe chosen such that it is a specific substrate for the serine proteaseplasmin. The C-terminal amino acid that is coupled to the spacerdrugmoiety is arginine or lysine, preferably an L-lysine residue. Plasmin isknown to cleave most easily after a lysine residue. The amino acid atthe N-terminus possesses the D-configuration in order to prevent in vivocleavage by ubiquitous amino peptidases. Protecting the N-terminal aminofunction by a Boc or Fmoc group can also prevent unwanted peptidasecleavage. The amino acid in the middle is preferably a hydrophobicL-amino acid and is selected from the group consisting of alanine,valine, leucine, isoleucine, methionine, phenylalanine,cyclohexylglycine, tryptophan and proline. Preferred tripeptidesequences are D-alanylphenylalanyllysine, D-valylleucyllysine,D-alanylleucyllysine, D-valylphenylalanyllysine,D-valyltryptophanyllysine, and D-alanyltryptophanyllysine.

By converting the two amino groups of the tripeptide into thecorresponding ammonium salts, the water solubility of the prodrug shouldbe improved.

In the present invention the synthesis and application of new elongatedspacer systems is described. In one embodiment, this spacerself-eliminates through a 1,(4+2n)-elimination process (FIGS. 4, 5).These 1,(4+2n)-elimination spacers are elongated with respect to theconventional 1,6-elimination spacer. Proof of principle of1,8-elimination was delivered upon chemical reduction of thenitrocinnamyl carbonate derivative of paclitaxel using zinc and aceticacid (FIGS. 6, 7). Released paclitaxel was isolated in good yield.Firstly, the doubly protected tripeptide was synthesized (FIG. 8). The1,8-elimination spacer itself was synthesized from 4-nitrocinnamylalcohol as depicted in FIG. 9. 4—Aminocinnamyl alcohol was incorporatedbetween doxorubicin and a tripeptide for plasmin activation (FIG. 10).What is also disclosed in this invention are prodrugs that contain twoor more electronic cascade spacers connected to one another,incorporated between specifier and drug (FIG. 11). Prodrugs containinglinker systems of this kind have not been reported before. Thisembodiment of the present invention was exemplified by synthesizing twoprodrugs containing a tripeptide specifier coupled to doxorubicin orpaclitaxel via two 1,6-elimination spacers. This protected tripeptidewas subsequently coupled with 4-aminobenzyl alcohol, and the resultingbenzylic alcohol was activated with 4-nitrophenyl chloroformate to yieldthe corresponding 4-nitrophenyl carbonate (FIG. 12). In a very efficientreaction a second molecule of 4-aminobenzyl alcohol was coupled to theactivated carbonate in which hydroxy benzotriazole HOBt) was employed asa catalyst to yield the corresponding tripeptide-double spacer conjugate(FIG. 13). When this reaction was performed using diphenyl phosphinicacid as a catalyst, the product was isolated in only 16 percent yield(FIG. 14). The peptide-double spacer conjugate was incorporated into adoxorubicin prodrug (FIG. 15) and a paclitaxel prodrug (FIG. 16), bysubsequent chloroformate activation, coupling with the drug and finaldeprotection. A double spacer-containing doxorubicin prodrug with atryptophan residue instead of phenylalanine was also synthesized (FIGS.17, 18). According to a further embodiment a third 4-aminobenzyl alcoholspacer was reacted with the 4-nitrophenyl carbonate activatedtripeptide-double spacer conjugate to yield the correspondingtripeptide-triple spacer conjugate (FIG. 19). This compound wassubsequently converted to the corresponding triple electronic cascadespacer containing doxorubicin prodrug employing a similar route asdepicted in FIGS. 15 and 16.

What is also claimed are prodrugs in which the promoiety is coupled to ahydroxyl group of parent moiety Z via a carbamate linkage. Thesecarbamate coupled prodrugs contain an elongated linker system thatcontains both one or more electronic cascade spacers and an ω-aminoaminocarbonyl cyclisation spacer (FIG. 20). Paclitaxel-2′-carbamateprodrugs of this type were synthesized via a novel convergent route;which leads to high yields, as claimed in claims 35 and 36. Paclitaxelwill be released after one or more 1,(4+2n)-eliminations (n=0,1,2,3,4,5,. . . 10) and a subsequent intramolecular cyclisation. In the presentinvention the cyclisation spacer is connected to the 2′-OH group ofpaclitaxel through a carbamate linkage (FIG. 20). Firstly, paclitaxelwas selectively activated at the 2′-position (FIG. 21) Secondly, amono-protected cyclisation spacer was coupled to the 2′-activatedpaclitaxel analog and the protective group was removed under acidicconditions to yield the first fragment (FIG. 22). The second fragmentwas synthesized by connecting the 1,6-elimination spacer to thetripeptide specifier and subsequent 4-nitrophenyl chloroformateactivation of the benzylic alcohol function (FIG. 12). Then, bothfragments were coupled to one another (FIG. 23) and the coupled productwas deprotected (FIG. 24). Coupling of two separate fragments accordingto this strategy in which in the final stage the chemical link betweenthe two spacers is established, did provide the most efficient route tothe paclitaxel prodrug. This is a novel route to obtain prodrugs of thistype, in which a specifier is connected to a hydroxyl containing drugvia an electronic cascade spacer system (connected to the specifier) anda cyclisation spacer (connected to the drug). The preparation of twoother prodrugs of paclitaxel that contain both an electronic cascadespacer system and a cyclisation spacer is depicted in FIGS. 25–27. InFIG. 28 previously reported plasmin-activatible prodrugs containing oneelectronic cascade spacer are depicted.

In a further aspect the invention relates to processes for the synthesisof the prodrugs as defined above. The invention e.g. relates to aprocess for the synthesis of prodrugs as defined above having at leastone electronic cascade spacer group, and an ω-amino aminocarbonylcyclisation elimination spacer group, connected to each other,incorporated between a specifier group and a drug molecule such thatsaid drug molecule is connected to said cyclisation elimination spacergroup, via the hydroxyl functionality of the drug molecule, by couplinga first electronic cascade spacer group, connected to said specifiergroup, if desired via at least one, second electronic cascade spacergroup, being the same or different as said first electronic cascadespacer group, to said cyclisation elimination spacer group. In apreferred process said drug molecule is paclitaxel, and in a first stepa cyclisation elimination spacer group is coupled to paclitaxel via its2′-hydroxyl group through a carbamate linkage via addition of the freespacer-amine to a 4-nitrophenyl carbonate activated drug, followed bydeprotection to obtain a first fragment consisting of a cyclisationspacer connected with paclitaxel, and in a second step one or more1,(4+2n) electronic cascade spacers, being the same or different,wherein n is an integer of 0 to 10, are coupled to a specifier group,subsequently activated to the corresponding 4-nitrophenyl carbonate,whereafter in a third step the fragments obtained in the first andsecond step are coupled to one another under basic reaction conditions.

The invention further relates to a process for the synthesis of prodrugsas defined above, in which electronic cascade spacers are connected toone another by coupling of the terminal alcohol group of an electroniccascade spacer to the aniline amino group of another electronic cascadespacer through a carbamate linkage by conversion of the alcohol group ofthe first-mentioned electronic cascade spacer to the corresponding4-nitrophenyl carbonate and reacting this molecule with the otherelectronic cascade spacer in the presence of a catalytic amount of1-hydroxybenzotriazole either in the presence or absence of base.

In yet another aspect the invention relates to the use of any of thecompounds defined above for the manufacture of a pharmaceuticalpreparation for the treatment of a mammal being in need thereof. Theinvention also relates to methods of treating a mammal being in needthereof, whereby the method comprises the administration of apharmaceutical composition to the mammal in a therapeutically effectivedose.

In a further aspect the invention relates to a process for preparing apharmaceutical composition containing a compound as defined above, toprovide a solid or a liquid formulation for administration orally,topically or by injection. Such a process at least comprises the step ofmixing the compound with a pharmaceutically acceptable carrier.

The invention also relates to pharmaceutical compositions comprising thecompounds of the invention as defined above. The compounds of theinvention may be administered in purified form together with apharmaceutical carrier as a pharmaceutical composition. The preferredform depends on the intended mode of administration and therapeutic ordiagnostic application. The pharmaceutical carrier can be anycompatible, nontoxic substance suitable to deliver the compounds of theinvention to the patient. Pharmaceutically acceptable carriers are wellknown in the art and include, for example, aqueous solutions such as(sterile) water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil or injectableorganic esters, alcohol, fats, waxes, and inert solids may be used asthe carrier. A pharmaceutically acceptable carrier may further containphysiologically acceptable compounds that act, e.g. to stabilise or toincrease the absorption of the compounds of the invention. Suchphysiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilisers or excipients. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the composition. Pharmaceuticallyacceptable adjuvants, buffering agents, dispersing agents, and the like,may also be incorporated into the pharmaceutical compositions.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain colouringand flavouring to increase patient acceptance.

The compounds of the invention are however preferably administeredparentally. Preparations of the compounds of the invention for parentaladministration must be sterile. Sterilisation is readily accomplished byfiltration through sterile filtration membranes, optionally prior to orfollowing lyophilisation and reconstitution. The parental route foradministration of compounds of the invention is in accord with knownmethods, e.g. injection or infusion by intravenous, intraperitoneal,intramuscular, intraarterial or intralesional routes. The compounds ofthe invention may be administered continuously by infusion or by bolusinjection. A typical composition for intravenous infusion could be madeup to contain 100 to 500 ml of sterile 0.9% NaCl or 5% glucoseoptionally supplemented with a 20% albumin solution and 1 mg to 10 g ofthe compound of the invention, depending on the particular type ofcompound of the invention and its required dosing regime. Methods forpreparing parenterally administrable compositions are well known in theart and described in more detail in various sources, including, forexample, Remington's Pharmaceutical Science (15th ed., Mack Publishing,Easton, Pa., 1980) (incorporated by reference in its entirety for allpurposes).

The invention also relates to compounds as defined above, wherein thespecifier V is removed by an enzyme that is transported to the vicinityof target cells or target tissue via antibody-directed enzyme prodrugtherapy (ADEPT), polymer-directed enzyme prodrug therapy (PDEPT),virus-directed enzyme prodrug therapy (VDEPT) or gene-directed enzymeprodrug therapy (GDEPT) (see e.g. U.S. Pat. No. 4,975,278, Melton etal., 1996, J. Natl. Can. Inst. 88(3/4):153–165).

The invention is further exemplified by the following Examples. Theseexamples are for illustrative purposes and are not intended to limit thescope of the invention

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows schematically the conversion of a spacer containingtripartate prodrug into the parent drug.

FIG. 2 shows schematically the structure of an elongated spacercontaining prodrug.

FIG. 3 shows the principle of 1,6-elimination.

FIG. 4 shows the principle of 1,8-elimination.

FIG. 5 shows the principle of 1,10-elimination.

FIG. 6 shows the synthesis of the model paclitaxel-containing compoundto prove the principle of 1,8-elimination.

FIG. 7 shows the mechanism for the release of paclitaxel after reductionand 1,8-elimination.

FIG. 8 shows the synthesis of the doubly Aloc-protected D-Ala-Phe-Lystripeptide.

FIG. 9 shows the synthesis of the 1,8-elimination spacerpara-aminocinnamyl alcohol (PACA).

FIG. 10 shows the synthesis of a 1,8-elimination spacer containingprodrug.

FIG. 11 shows schematically the structure of a prodrug containing two ormore electronic cascade spacers.

FIG. 12 shows the synthesis of para-nitrophenyl (PNP)carbonate-activated tripeptide-spacer conjugate.

FIG. 13 shows the catalytic coupling of a second electronic cascadespacer molecule (para-aminobenzyl alcohol (PABA)) to the 4-nitrophenylcarbonate-activated tripeptide-spacer conjugate in the presence ofhydroxy benzotriazole (HOBt).

FIG. 14 shows a reaction to chemically link two electronic cascadespacer molecules by coupling a second electronic cascade 1,6-eliminationspacer molecule to a 4-nitrophenyl carbonate activatedtripeptide-1,6-elimination spacer conjugate in the presence of catalyticamounts of diphenyl phosphinic acid.

FIG. 15 shows the synthesis of a doxorubicin containing double1,6-elimination spacer containing prodrug.

FIG. 16 shows the synthesis of a paclitaxel containing double1,6-elimination spacer containing prodrug.

FIG. 17 shows the synthesis of a tryptophan-containing tripeptide doublespacer conjugate.

FIG. 18 shows the synthesis of a tryptophan-containing doxorubicinprodrug.

FIG. 19 shows the synthesis of a doxorubicin containing triple1,6-elimination spacer containing prodrug.

FIG. 20 shows schematically the structure of a prodrug of paclitaxelthat contains both an electronic cascade spacer and a cyclisation spacercoupled to the drug via a 2′-carbamate linkage.

FIG. 21 shows the regioselective synthesis of 2′-(4nitrophenylcarbonate) activated paclitaxel using 4-nitrophenyl chloroformate at lowtemperature.

FIG. 22 shows the synthesis of the acid protected paclitaxel-ω-aminoaminocarbonyl cyclisation spacer conjugate.

FIG. 23 shows the coupling of the acid protected paclitaxel-ω-aminoaminocarbonyl cyclisation spacer conjugate to the 4-nitrophenylcarbonate activated tripeptide-1,6-elimination spacer conjugate.

FIG. 24 shows the deprotection reaction to obtain the paclitaxel prodrugthat contains a 1,6-elimination spacer and an ω-amino aminocarbonylcyclisation spacer.

FIG. 25 shows the preparation of a paclitaxel prodrug that contains two1,6-elimination spacers and an ω-amino aminocarbonyl cyclisation spacer.

FIG. 26 shows the synthesis of para-nitrophenyl (PNP)carbonate-activated doubly para-nitrobenzyloxycarbonyl-protectedtripeptide-spacer conjugate.

FIG. 27 shows the preparation of a paclitaxel prodrug that contains a1,8-elimination spacer and an ω-amino aminocarbonyl cyclisation spacer.

FIG. 28 shows the structure of previously reported doxorubicin andpaclitaxel prodrugs containing one electronic cascade spacer.

EXAMPLES Example 1

Synthesis of 2′-[4-nitrocinnamyl carbonate]-paclitaxel 1.

To a solution of 200 mg (1.12 mmol, 4.8 equiv) 4-nitrocinnamyl alcoholin dry dichloromethane/tetrahydrofuran under an Argon atmosphere wasadded pyridine (94 μl, 5.0 equiv) and 4-nitrophenyl chloroformate (236mg, 5.0 equiv). The reaction mixture was stirred for 12 h at roomtemperature. The mixture was cooled to 0° C. and a catalytic amount ofDMAP, a few drops of triethyl amine and 200 mg paclitaxel (1.0 equiv)were added. The reaction mixture was stirred at room temperature for 12h. Solvents were evaporated and the remaining solid was dissolved indichlorometaane. The organic layer was thoroughly washed with asaturated sodium bicarbonate solution, 0.5 N potassium bisulfate andbrine and dried over anhydrous sodium sulfate. After evaporation of thesolvents the residual yellow oil was purified by means of columnchromatography (ethyl acetate-hexane; 1:1), to yield 144 mg of 1 (58%).M.P. 151° C.; ¹H-NMR (300 Mz, CDCl₃) δ 1.17 (s, 3H, 17), 1.22 (s, 3H,16), 1.70 (s, 3H, 19), 1.96 (s, 3H, 18), 2.22 (s, 3H, 10-OAc), 2.46 (s,3H, 4-OAc), 2.55 (m, 1H, 6a), 3.82 (d, 1H, J=7.0 Hz, 3), 4.26 (d, 1H,J=8.4 Hz, 20b), 4.32 (d, 1H, J=8.4 Hz, 20a), 4.39 (m, 1H, 7), 4.87 (bt,2H, CH₂-spacer), 4.99 (bd, 1H, J=7.9 Hz, 5), 5.46 (d, 1H, J=2.8 Hz, 2′),5.72 (d, 1H, J=7.1 Hz, 2), 6.01 (m, 1H, 3′), 6.26 (bt, 1H, 13), 6.34 (s,1H, 10), 6.43 (dt, 1H, J=16.0 Hz, HC═CH—CH₂), 6.75 (d, 1H, J=16.0 Hz,HC═CH—CH₂), 7.35–7.67 (m, 13H, aromatic), 7.75 (d, 2H, J=7.2 Hz,aromatic), 8.15 (d, 2H, J=7.2 Hz, aromatic), 8.19 (d, 2H, J=8.7 Hz,nitrophenyl) ppm; MS (FAB) m/e 1059 (M+H)⁺, 1081 (M+Na)⁺; Anal.(C₅₇H₅₈N₂O₁₈.2½H₂O) calculated C, 62.01%; H, 5.75%; N, 2.54%; measuredC, 62.06%; H, 5.31%; N, 2.60%.

Example 2

Principle of 1,8-elimination: Chemical Reduction of the NitrocinnamylCarbonate 1.

36 mg of 2′-[nitrocinnamyl carbonate]-paclitaxel 1 was dissolved in 8 mlmethanol and 2 ml acetic acid. A catalytic amount of zinc powder wasadded and the red mixture was stirred for 12 h. Dichloromethane wasadded and the organic layer was washed with saturated sodiumbicarbonate, 0.5 N potassium bisulfate, brine, and water and dried overanhydrous sodium sulfate. After evaporation of the solvents the residualyellow film was purified by means of column chromatography (ethylacetate-hexane; 2:1), to yield 28 mg of paclitaxel (confirmation by 300MHz ¹H-NMR) and 4 mg of unreacted starting compound. When the compoundwas stirred in the absence of zinc powder under the same conditions, nopaclitaxel was formed, indicating that reduction of the nitro group byzinc leads to the release of paclitaxel.

Example 3

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-OH 9.

Step a: Synthesis of Fmoc-Phe-Lys(Boc)-OBu 4.

To a solution of 2.50 g Fmoc-Phe-ONSu 2 (ONSu=N-hydroxysuccinimide)(5.16 mmol) in dry dichloromethane under an Argon atmosphere were addedat 0° C. 0.791 ml triethyl amine (1.1 eq.) and 1.92 g H-Lys(Boc)OBu.HCl3 (1.1 eq.). The reaction mixture was stirred at room temperature for 5hours, then dichloromethane was added and the organic layer was washedwith 10% citric acid, saturated sodium bicarbonate and water. Theorganic layer was dried over anhydrous sodium sulphate and evaporated.The resulting white solid 4 (3.08 g, 89%) was used without furtherpurification. M.P. 93° C.; ¹H-NMR (300 MHz, CDCl₃): δ 1.10–1.90 (m, 24H,6 CH₂-Lys and 18 tert-butyl), 3.06 (m, 2H, N—CH₂-Lys and benzylic), 4.19(t, 1H, Fmoc), 4.25–4.55 (m, 4H, 2 Fmoc and 2 Hα), 7.19–7.78 (m, 13H,aromatic) ppm; MS (FAB) m/e 672 (M+H)⁺, 694 (M+Na)⁺; C₃₉H₄₉N₃O₇calculated C, 69.72%; H, 7.35%; N, 6.25%; measured C, 69.69%; H, 7.48%;N, 6.22%.

Step b: Synthesis of Boc-D-Ala-Phe-Lys(Doc)OBu 7.

3.08 g (4.58 mmol) of Fmoc-Phe-Lys(Boc)OBu 4 was dissolved in 100 ml ofdioxane/methanol/2N sodium hydroxide (70/25/5) and stirred at roomtemperature for approximately 1 hour. The reaction mixture wasneutralised with acetic acid (0.571 ml) and organic solvents wereevaporated. Water and dioxane was added and the solution was freezedried. Diisopropylether was added to the resulting solid. Afterfiltration, the filtrate was evaporated. The residual product 5 wasdissolved in dry dichloromethane and added at 0° C. to a solution of1.19 g (4.16 mmol) Boc-D-Ala-ONSu 6 and 0.634 ml (1.1 eq.) of triethylamine in dry dichloromethane. The reaction mixture was stirred overnightafter which dichloromethane was added. The organic layer was washed with10% citric acid, saturated sodium bicarbonate and water. The organiclayer was dried over anhydrous sodium sulphate and evaporated. Theproduct was purified by means of column chromatography (SiO₂—CHCl₃/MeOH20/1) to afford 2.56 g (4.13 mmol. 99%) of Boc-D-Ala-Phe-Lys(Boc)-OBu 7as a white foam. M.P. 59° C.; ¹H-NMR (300 MHz, CDCl₃): δ 1.25 (d, 3H,CH₃-Ala), 1.43 (bs, 27H, tert-butyl), 1.00–1.90 (m, 6H, CH₂-Lys).2.80–3.30 (m, 4H, N—CH₂-Lys and benzylic), 4.15 (m, 1H, Hα), 4.35 (m,1H, Hα), 4.64 (dd, 1H, Hα), 7.15–7.35 (m, 5H, aromatic) ppm; MS (FAB)m/e 621 (M+H)⁺, 643 (M+Na)⁺; C₃₂H₅₂N₄O₈ (.1/2H₂O) calculated C, 61.03%;H, 8.48%; N, 8.90%; measured C, 61.15%; H, 8.44%; N, 8.66%.

Step c: Synthesis of D-Ala-Phe-Lys-OH 8.

2.56 g (4.13 mmol) Boc-D-Ala-Phe-Lys(Boc)-OBu 7 was stirred in asolution of HCl in EtOAc (3M). After 5 hours the solvent was evaporated,tert-butanol was added and evaporated twice to remove remaininghydrochloric acid. The resulting product was freeze dried in a mixtureof dioxane/water to yield a cream coloured powder 8, which was usedwithout further purification. ¹H-NMR (300 MHz, D₂O): δ 0.94 (d, 3H,CH₃-Ala), 1.10–1.85 (m, 6H, CH₂-Lys), (dd, 1H, Hα), 4.54 (q, 1H, Hα),7.10–7.22 (m, 5H, aromatic) ppm; MS (FAB) m/e 365 (M+H)⁺.

Step d: Synthesis of Aloc-D-Ala-Phe-Lvs(Aloc)-OH 9.

To solution of 706 mg (1.61 mmol) D-Ala-Phe-Lys-OH 8 inwater/acetonitrile was added triethyl amine until a pH of 9–9.5 wasreached. Then a solution of 704 mg (2.2 eq.) Aloc-ONSu in acetonitrilewas added and the reaction mixture was kept basic by adding triethylamine. After the pH of the mixture did not alter anymore, a 0.5 Msolution of HCl was added until a pH of 3 was reached. The mixture wasthoroughly extracted with dichloromethane. The organic layer was washedwith water and the water layer was extracted again with dichloromethane.The organic layer was dried over anhydrous sodium sulphate andevaporated to dryness to result in the desired product 9 as a creamcoloured foam (742 mg, 86%). M.P. 141° C.; ¹H-NMR (300 MHz, CDCl₃): δ1.10–1.95 (m, 6H, CH₂-Lys), 1.21 (d, 3H, CH₂-Ala), 2.90–3.30 (m, 4H,N—CH₂-Lys and benzylic), 4.20 (m, 1H, Hα), 4.55 (m, 5H, Hα and 4 Aloc),4.76 (bd, 1H, Hα), 5.17–5.31 (m, 4H, Aloc), 5.83–5.92 (m, 2H, Aloc),7.20–7.28 (m, 5H, aromatic) ppm; MS (FAB) m/e 533 (M+H)⁺, 555 (M+Na)⁺;C₂₆H₃₆N₄O₈ calculated C 58.63%; H, 6.81%; N, 10.52%; measured C, 58.54%;H, 6.81%; N, 10.28%.

Example 4

Synthesis of 4-aminocinnamyl Alcohol 10.

To a solution of 1.5 g (8.37 mmol) of 4-nitrocinnamyl alcohol inTHF/methanol (60 mL, 1:1 v/v) was added a catalytic amount of RaneyNickel and hydrazine monohydrate (1.22 mL, 25.1 mmol). The mixture wasstirred at room temperature for 3 h, additional hydrazine monohydrate(1.22 mL) being added after 1.5 h. The reaction mixture was filteredover HFLO and concentrated under reduced pressure to 5 mL.Dichloromethane (100 mL) was added and the resultant solution was washedwith water, dried over Na₂SO₄, filtered, and concentrated, which gave 10(1.24 g, 8.31 mmol, 99%).

¹H NMR (300 MHz, CDCl₃) δ 4.24 (d, 2H, J=6.1 Hz, CH ₂OH), 6.11–6.20 (dt,1H, J=6.1 Hz, J=15.8 Hz, CH═CH—CH₂OH), 6.48 (d, 1H, J=15.8 Hz,CH═CH—CH₂), 6.62 (d, 2H, J=11.1 Hz, aromatic), 7.19 (d, 2H, J=11.0 Hz,aromatic) ppm; MS (EI) m/e 149(M)⁺.

Example 5

Synthesis of Fmoc-D-Ala-Phe-Lys(Emoc)PACA 11.

To a solution of D-Ala-Phe-Lys-OH 8 (3.20 g, 8.79 mmol) in a 7:3 mixtureof water and acetonitrile (300 mL) was added triethylamine until a pH of8.5 was reached. A solution of Fmoc-OSu (5.93 g, 17.6 mmol) inacetonitrile (50 ml) was added. The pH of the resultant solution waskept at a pH of 8.5–9.0 by the addition of triethylamine. When the pHdid no longer change, a 1 N aqueous HCl solution was added to neutralizethe solution. The solution was concentrated under reduced pressure toremove acetonitrile. The resultant aqueous solution was extracted 4times with ethyl acetate. The combined organic layers were dried overNa₂SO₄, filtered, and concentrated under reduced pressure. The residuewas thoroughly washed with diisopropyl ether to remove apolarcontaminations. This gave 5.54 g (6.85 mmol, 78%) of crude 9b.

A solution of 9b (500 mg, 0.618 mmol) in THF (50 mL) was cooled to −40°C. Then, N-methylmorpholine (75 μL, 0.68 mmol) and isobutylchloroformate (89 μL, 0.68 mmol) were added consecutively. The resultantsolution was stirred at −40° C. for 3.5 h. A solution ofpara-aminocinnamyl alcohol (111 mg, 0.742 mmol) in THF (20 mL) was addedslowly. The reaction mixture was stirred at −20° C. for 5 h and thenconcentrated under reduced pressure. The crude product was purified bymeans of column chromatography (SiO₂—CH₂Cl₂/MeOH 93/7). This gave 516 mgof 11 (0.549 mmol, 89%).

¹H NMR (300 MF CDCl₃/CD₃OD) δ 1.17 (d, 3H, J=6.8 Hz, CH₃ of Ala),1.20–2.00 (m, 6H, 3×CH₂-Lys), 2.89–3.26 (m, 4H, N—CH₂ of Lys and Ph-CH₂of Phe), 3.88–4.58 (m, 11H, CH₂ and CH of Fmoc and CH₂ of spacer and3×Hα), 6.21 (dt, 1H, J=15.9 Hz, J=5.8 Hz, CH═CH—CH ₂), 6.48 (d, 1H,J=15.7 Hz, CH═CH—CH₂) 7.15–7.43 (m, 17H, aromatic), 7.52–7.57 (m, 2H,aromatic), 7.67 (d, 1H, J=7.4 Hz, aromatic), 7.71 (d, 1H, J=7.4 Hz,aromatic) ppm; MS (FAB) m/e 940 (M+H)⁺.

Example 6

Synthesis of Fmoc-D-Ala-Phe-Lys(Fmoc)-PACC-PNP 12.

To a solution of 11 (800 mg, 0.851 mmol) in THF (15 mL) were added at 0°C. DIPEA (594 μL, 3.40 mmol), para-nitrophenyl chloroformate (515 mg,2.55 mmol), and pyridine (17.3 μL, 0.213 mmol). The reaction mixture wasstirred at room temperature for 2 h, after which dichloromethane (50 mL)and water (50 mL) were added. The aqueous layer was separated from theorganic layer and extracted with dichloromethane (3×100 mL). Thecombined organic layers were washed with water, a saturated aqueousNaHCO₃ solution, and brine, dried with anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure. The residue was washed with a 1:2mixture of dichloromethane and diethyl ether. This gave 12 (812 mg,0.734 mmol, 86%).

¹H NMR (300 MHz, DMSO-d₄) δ 0.95 (d, 3H, J=7.8 Hz, CH₃ of Ala),1.05–1.90 (m, 6H, 3×CH₂ of Lys), 2.90 (dd, 1H, J=11.4 Hz, J=15.0 Hz, CH₂of Phe), 3.11 (m, 2H, N—CH₂ of Lys), 3.26 (m, 1H, CH₂ of Phe), 4.24–4.89(m, 9H, CH₂ and CH of Fmoc and 3×Hα), 5.24 (d, 2H, J=6.6 Hz, CH═CH—CH₂), 6.81 (dt, 1H, J=17.4 Hz, J=7.0 Hz, CH═CH—CH₂), 7.23 (d, 1H, J=17.4Hz, CH═CH—CH₂), 7.66–8.25 (m, 23H, aromatic), 8.44–8.48 (m, 4H,aromatic), 8.97 (d, 2H, J=10.1 Hz, aromatic) ppm; MS (FAB) m/e 1105(M+H)⁺.

Example 7

Synthesis of Fmoc-D-Ala-Phe-Lys(fmoc)-PACC-DOX 13.

To a solution of 12 (100 mg, 90.5 μmol) in N-methylpyrrolidinone (2 mL)were added triethylamine (13.2 μL, 95.0 μmol) and doxorubicinhydrochloride (55.1 mg, 95.0 μmol). The reaction mixture was stirred atroom temperature for 15 h and then poured into 10% isopropanol in ethylacetate (25 mL). The resultant solution was washed with water, dilutedwith dichloromethane (50 mL), dried with anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure. The crude product was purified bycolumn chromatography (SiO₂—CH₂Cl₂/MeOH 93/7) and subsequentprecipitation from diethyl ether, which gave 13 (82.3 mg, 54.5 μmol,60%).

¹H NMR (300 MHz, DMSO-d₆) δ 0.98 (d, 3H, J=6.8 Hz, CH₃ of Ala), 1.12 (d,3H, J=6.7 Hz, 5′-Me), 1.24–2.25 (m, 8H, 3×CH₂ of Lys and 2′ and 8), 2.79(m, 1H, CH₂ of Phe), 2.97 (m, 4H, N—CH₂ of Lys and 10), 3.11 (m, 1H, CH₂of Phe), 3.47 (m, 1H, 4′), 3.74 (m, 1H, 3′), 3.97 (s, 3H, OCH₃),3.97–4.37 & 4.54–4.60 & 4.72 & 4.85 & 4.97 & 5.24 & 5.47 (14H, CH₂ andCH of Fmoc and CH═CH—CH ₂ and 1′ and 5′ and 7 and 14 and OH), 6.20 (m,1H, CH═CH—CH₂), 6.55 (d, 1H, J=16.2 Hz, CH═CH—CH₂), 7.14–7.93 (m, 32H,aromatic) ppm; MS (FAB) m/e 1533 (M+Na)⁺.

Example 8

Synthesis of D-Ala-Phe-Lys-PACC-DOX (.2HCl) 14.

To a stirred solution of 50 (40.0 mg, 26.5 μmol) in DMF (2 mL) was addedpiperidine (128 μL, 1.30 mmol). After 10 min, the reaction mixture wasslowly added to a stirred solution of ice-cold diethyl ether. Theprecipitate was collected by means of centrifugation, washed withdiethyl ether, and dissolved in ethyl acetate. An approximately 0.5 Msolution of HCl in ethyl acetate (200 μL) was added and the precipitateformed was collected by means of centrifugation. The residue wasdissolved in a 2:1 mixture of tert-butanol and chloroform, and theresultant solution was concentrated under reduced pressure. Thisprocedure was repeated twice, yielding 51 (29.4 mg, 25.8 μmol, 97%) asan orange solid after freeze-drying.

¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 1.22 (d, 3H, 5′-Me), 1.29 (d, 3H, CH₃ ofAla), 1.30–2.00 (m, 8H, 3×CH₂ of Lys and 2′), 2.17 (br.d, 1H, 8), 2.39(br.d, 1H, 8), 2.93–2,35 (m, 7H, CH₂ of Phe and N—CH₂ of Lys and 10 and4′), 3.63 (m, 1H, 3′), 3.85–4.20 (m, 3H, 5′ and 2×Hα), 4.07 (s, 3H,OCH₃), 4.50–4.78 (m, 5H, Hα and 14 and CH═CH—CH ₂), 5.25 (m, 1H, 1′),5.48 (m, 1H, 7), 6.15 (m, 1H, CH═CH—CH₂), 6.56 (d, 1H, J=14.6 Hz,CH═CH—CH₂), 7.23–7.56 (m, 10H, aromatic and 3), 7.82 (t, 1H, J=8.0 Hz,2), 8.01 (m, 1H, 1) ppm; MS (FAB) m/e 1065 (M+H)⁺.

Example 9

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABA 15.

A solution of 730 mg (1.37 mmol) protected tripeptideAloc-D-Ala-Phe-Lys(Aloc)-OH 9 was dissolved in dry THF under an Argonatmosphere and cooled to −40° C. NMM (166 μl, 1.1 eq.) and isobutylchloroformate (196 μl, 1.1 eq.) were added. The reaction mixture wasstirred for 3 hours at a temperature below −30° C. A solution of4-aminobenzyl alcohol (203 mg, 1.2 eq.) and NMM (181 μl, 1.2 eq.) in dryTHF was added dropwise to the reaction mixture. After 2 hours THF wasevaporated and dichloromethane was added. The organic layer was washedwith saturated sodium bicarbonate, a 0.5 N potassium bisulphate solutionand brine, dried over anhydrous sodium sulphate, and evaporated. Theresidual pale yellow solid was purified by means of columnchromatography (SiO₂—CHCl₃/MeOH 9/1) to afford 812 mg (93%) of thedesired product 15 as a cream coloured powder. M.P. 156° C.; ¹H-NMR (300MHz, DMSO-D⁶): δ 0.96 (d, 3H, CH₃-Ala), 1.10–1.85 (m, 6H, CH₂-Lys), 2.77(dd, 1H, benzylic Phe), 2.97 (bd, 2H, N—CH₂-Lys), 3.09 (dd, 1H, benzylicPhe), 4.00 (t, 1H, Hα), 4.20–4.60 (m, 8H, 2 Hα and 4 Aloc and CH ₂—OH),5.00–5.35 (m, 4H, Aloc), 5.76–5.95 (m, 2H, Aloc), 7.05–7.30 (m, 7H,aromatic), 7.41 (d, 1H, NH), 7.56 (d, 2H, aromatic), 8.12 (d, 1H, NH),8.18 (d, 1H, NH), 9.80 (s, 1H, NH anilide) ppm; MS (FAB) m/e 638 (M+H)⁺,660 (M+Na)⁺; C₃₃H₄₃N₅O₈ (.½ H₂O) calculated C, 61.29%; H, 6.86%; N,10.83%; measured C, 61.39%; H, 6.54%; N, 10.55%.

Example 10

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PNP 16.

To a solution of 384 mg (0.602 mmol) 15 in dry TBF/CH₂Cl₂ under an Argonatmosphere, 4-nitrophenyl chloroformate (182 mg, 1.5 eq.) and drypyridine (73 μl, 1.5 eq.) were added. The reaction mixture was stirredat room temperature for 48 hours, then EtOAc was added. The organiclayer was washed with 10% citric acid, brine and water, dried overanhydrous sodium sulphate and evaporated yielding a yellow solid. Theproduct was purified by means of column chromatography (SiO₂—CH₂Cl₂/MeOH30/1) to afford 324 mg (67%) of carbonate 16. ¹H-NMR (300 MHz,CDCl₃/CD₃OD): δ 1.21 (d, 3H, CH₃-Ala), 1.25–2.05 (m, 6H, CH₂-Lys), 2.95(dd, 1H, benzylic Phe), 3.13 (bt, 2H, N—CH₂-Lys), 3.27 (dd, 1H, benzylicPhe), 4.08 (dd, 1H, Hα), 4.25 (dd, 1H, Hα), 4.30–4.65 (m, 5H, Hα and 4Aloc), 5.04–5.35 (m, 4H, Aloc), 5.26 (s, 2H, CH ₂—OH), 5.65–6.00 (m, 2H,Aloc), 7.10–7.35 (m, 5H, aromatic), 7.39–7.43 (2*d, 4H, aromatic), 7.71(d, 2H, aromatic), 8.28 (d, 2H, aromatic) ppm; MS (FAB) m/e 803 (M+H)⁺,825 (M+Na)⁺.

Example 11

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABA 17.

To a solution of 156 mg (194 μmol) of compound 16 and 26.3 mg (1.1 eq.)PABA in dry N,N-dimethyl formamide under an Argon atmosphere was addeddiisopropylethyl amine (34 μl, 1.0 eq.) and a catalytic amount ofN-hydroxybenzotriazole (7.9 mg, 0.3 eq.). The reaction solution wasstirred for 24 hours after which it was diluted with 10%propanol-2/EtOAc. The organic layer was washed with saturated sodiumbicarbonate, 0.5 N potassium hydrogensulfate and brine, dried overanhydrous sodium sulfate and evaporated to dryness. The yellow residualfilm was purified by means of column chromatography (SiO₂—CHCl₃/MeOH9/1) to yield 148 mg (97%) of the desired product 17. M.P. 196–197° C.;¹H-NMR (300 MHz, CDCl₃): δ 1.20 (d, 3H, ³J=6.4 Hz, CH₃-Ala), 1.27–2.05(m, 6H, 3 CH₂-Lys), 2.99 (dd, 1H, benzylic), 3.14 (m, 2H, N—CH₂-Lys),3.27 (dd, 1H, benzylic), 4.00–4.64 (m, 7H, 3 Hα and Aloc), 4.57 (s, 2H,benzylic-spacer), 5.14 (s, 2H, benzylic-spacer), 5.06–5.37 (m, 4H,Aloc), 5.72 (m, 1H, Aloc), 5.88 (m, 1H, Aloc), 7.10–7.46 (m, 11H,aromatic), 7.64 (d, 2H, ³J=8.3 Hz, aromatic) ppm; MS (FAB) m/e 809(M+Na)⁺;; C₄₁H₅₀N₆O₁₀ (.½H₂O) calculated C 61.87%; H, 6.46%; N, 10.56%;measured C, 61.84%; H, 6.38%; N, 10.38%.

Example 12

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-PNP 18.

A solution of 80.2 mg (102 μmol) of compound 17, pyridine (25 μl, 3.0eq.) and 4-nitrophenyl chloroformate (44.3 mg, 220 μmol) in drytetrahydrofuran/dichloromethane was stirred under an Argon atmosphere at0° C. for two hours and overnight at room temperature. The solution wasevaporated in vacuo and the residual product was dissolved indichloromethane. After washing the organic layer with brine and 0.5 Npotassium bisulfate, the organic layer was dried over anhydrous sodiumsulfate and concentrated to dryness. The resulting crude product wassubjected to column chromatography (SiO₂—CHCl₃/MeOH 20/1) to obtain 61.9mg (84%) of compound 18. M.P. 69–70° C.; ¹H-NMR (300 MHz, CDCl₃/CD₃OD):δ 1.23 (d, 3H, ³J=7.0 Hz, CH₃-Ala), 1.10–2.08 (m, 6H, 3 CH₂-Lys), 3.04(m, 1H, benzylic), 3.13 (m, 2H, N—CH₂-Lys), 3.27 (bd, 1H, benzylic),4.06 (m, 1H, Hα), 4.26 (m, 1H, Hα), 4.35–4.70 (m, 5H, Hα and Aloc),5.04–5.47 (m, 4H, Aloc), 5.14 (s, 2H, benzylic-spacer), 5.24 (s, 2H,benzylic-spacer), 5.72 (m, 1H, Aloc), 5.90 (m, 1H, Aloc), 7.10–7.46 (m,13H, aromatic), 7.65 (d, 2H, ³J=8.3 Hz, aromatic), 8.27 (d, 2H, ³J=9.1Hz, aromatic-PNP) ppm; MS (FAB) m/e 952 (M+H)⁺, 974 (M+Na)⁺; C₄₀H₄₆O₁₂(.¼H₂O) calculated C, 59.51%; H, 5.81%; N, 10.41%; measured C 59.52%; H,5.54%; N, 10.12%.

Example 13

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-DOX 19.

The double spacer containing 4-nitrophenyl carbonate 18 (140 mg, 0.147mmol) and doxorubicin-HCl (94.1 mg, 1.1 eq.) in N-methylpyrrolidinonewere treated at room temperature with triethyl amine (22.5 μl, 1.1 eq.).The reaction mixture was stirred in the dark for 72 hours, againtriethyl amine (1.1 eq.) was added and after an additional 24 hours thereaction mixture was diluted with 10% 2-propanol/ethyl acetate. Theorganic layer was washed with water and brine, and was dried (Na2SO₄).After evaporation of the solvents the crude product was purified bymeans of column chromatography (chloroform-methanol; 9:1) followed bycircular chromatography using a chromatotron supplied with a 2 mm silicaplate (chloroform-methanol; 9:1), to yield 72 mg (36%) of protectedprodrug 19. M.P. 129° C.; ¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 1.22 (d, 3H,J=7.1 Hz, sugar CH₃), 1.27 (d, 3H, J=6.7 Hz, CH₃-Ala), 1.25–2.00 (m, 8H,CH₂-Lys and 2′), 2.15 (dd, 1H, 8), 2.36 (bd, 1H, 8), 3.04 (bd, 1H,J=18.7 Hz, 10), 2.90–3.50 (m, 5H, benzylic Phe and N—CH₂-Lys and 10),3.37 (bs, 1H, 4′), 3.58 (m, 1H, 3′), 3.85 (m, 1H, Hα), 4.08 (s, 3H,OMe), 4.14 (m, 1H, Hα), 4.29 (dd, 1H, 5′), 4.3–74.68 (m, 5H, Hα and 4Aloc), 4.76 (s, 2H, 14), 4.96 (s, 2H, benzylic spacer), 5.11 (s, 2H,benzylic spacer), 5.02–5.40 (m, 4H, Aloc), 5.48 (bs, 1H, 1′), 5.61–6.00(m, 3H, Aloc and 7), 7.08–7.39 (m, 9H, aromatic 5H Phe and 4H spacers),7.33 (d, 2H, J=8.3 Hz, 2H aromatic spacer), 7.42 (d, 1H, J=8.4 Hz, 3),7.62 (d, 2H, J=8.0 Hz, 2H aromatic spacer), 7.80 (t, 1H, J=8.1 Hz, 2),8.03 (d, 1H, J=7.5 Hz, 1) ppm; MS (FAB) m/e 1378 (M+Na)⁺; Anal.(C₆₉H₇₇N₇O₂₂.2H₂O) calculated C, 59.52%; H, 5.86%; N, 7.04%; measured C,59.34%; H, 5.71%; N, 6.66%.

Example 14

Synthesis of H-D-Ala-Phe-Lys-PABC-PABC-DOX.5.7HCl 20.

To a solution of 48 mg (0.035 mmol) protected prodrug 19 in drytetrahydrofuran/dichloromethane under an argon atmosphere was addedmorpholine (31 μl, 10 eq.) together with a catalytic amount ofPd(PPh₃)₄. The reaction mixture was stirred for one hour in the dark.The red precipitate was collected by means of centrifugation. Ethylacetate was added and the mixture was acidified using 1.0 ml of 0.5 Mhydrochloric acid/ethyl acetate. The precipitate was collected by meansof centrifugation and washed several times with ethyl acetate.Tert-butanol was added and evaporated and the resulting red film wasfreeze dried in water yielding 37 mg (83%) of prodrug 20. Mp>300° C.;¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 1.20 (d, 3H, J=7.0 Hz, sugar CH₃), 1.27(d, 3H, J=6.5 Hz, CH₃-Ala), 1.38–2.05 (m, 8H, CH₂-Lys and 2′), 2.18 (dd,1H, 8), 2.36 (bd, 1H, 8), 2.82–3.41 (m, 6H, benzylic Phe and N—CH₂-Lysand 10), 3.37 (s, 1H, 4′), 3.60 (bs, 1H, 3′), 4.02 (m, 1H, Hα), 4.08 (s,3H, OMe), 4.18 (1,1H, Hα), 4.53 (dd, 1H, 5′), 4.66 (dd, 1H, Hα), 4.77(s, 2H, 14), 4.95 (bs, 2H, benzylic spacer), 5.14 (s, 2H, benzylicspacer), 5.27 (bs, 1H, 1′), 5.48 (bs, 1H, 7), 7.09–7.50 (m, 11H,aromatic 5H Phe and 6H spacers and 3), 7.58 (d, 2H, J=8.4 Hz, 2Haromatic spacer), 7.82 (t, 1H, J=8.0 Hz, 2), 8.03 (d, 1H, J=7.6 Hz, 1)ppm; MS (FAB) m/e 1188 (M+H)⁺, m/e 1210 (M+Na)⁺; Anal. (duplo)(C₆₁H₆₉N₇O₁₈.5.7HCl) calculated C, 52.42%; H, 5.39%; N, 7.01%; measuredC, 52.38%; H 5.71%; N, 7.14%.

Example 15

Synthesis of 2′-[Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC]-paclitaxel 21.

4-Nitrophenyl carbonate 18 (47.4 mg, 49.8 μmol) and paclitaxel (42.3 mg,1.0 eq.) in dry tetrahydrofuran/dichloromethane under an Argonatmosphere were treated at room temperature withN,N-dimethyl-4-aminopyridine (DMAP) (6.7 mg, 1.1 eq.). The reactionmixture was stirred in the dark for 48 hours and was then concentratedto dryness. The product was dissolved in dichloromethane and the organiclayer was washed with saturated sodium bicarbonate, 0.5 N potassiumbisulfate and brine and dried over anhydrous sodium sulfate. Afterevaporation of the solvents the residual yellow film was purified bymeans of column chromatography (SiO₂—EtOAc/Hex/MeOH 5/5/1), to yield67.5 mg (82%) of the desired protected paclitaxel prodrug 21. M.P.137–138° C.; ¹H-NMR (300 Mz, CDCl₃): δ 1.14 (s, 17), 1.23 (s, 3H, 16),1.27 (d, 3H, ³J=7.1 Hz, CH₃-Ala), 1.05–2.10 (m, 6H, CH₂-Lys), 1.67 (s,3H, 19), 1.89 (s, 3H, 18), 2.22 (s, 3H, 10-OAc), 2.44 (s, 3H, 4-OAc),2.97 (m, 1H, benzylic), 3.14 (m, 2H, N—CH₂-Lys), 3.21 (m, 1H, benzylic),3.81 (d, 1H, ³J=7.0 Hz, 3), 4.03 (m, 1H, Hα), 4.20 (d, 1H, ²J=8.4 Hz,20b), 4.31 (d, 1H, ²J=8.4 Hz, 20a), 4.43 (m, 1H, 7), 4.34–4.74 (m, 6H,Hα and Aloc), 4.90–5.37 (m, 11H, 2 Hα, Aloc, 5 and 2 benzylic-spacer),5.44 (d, 1H, ³J=2.9 Hz, 2′), 5.63 (m, 1H, Aloc), 5.69 (d, 1H, ³J=7.1 Hz,2), 5.87 (m, 1H, Aloc), 5.97 (bd, 1H, ³J=2.9 Hz, ³J=9.2 Hz, 3′), 6.26(m, 1H, 13), 6.29 (m, 1H, 10), 7.05–7.80 (m, 26H, aromatic), 8.14 (d,2H, 3J=7.2 Hz, aromatic) ppm; MS (FAB) m/e 1668 (M+H)⁺, 1689 (M+Na)⁺;C₈₉H₉₉N₇O₂₅ (.2H₂O) calculated C, 62.78%; H, 6.10%; N, 5.76%; measuredC, 62.55%; H, 5.82%; N, 5.57%.

Example 16

Synthesis of 2′-[H-D-Ala-Phe-Lys-PABC-PABC]-paclitaxel (.2HCl) 22.

To a solution of 51.4 mg (30.8 μmol) protected prodrug 21 in drytetrahydrofuran under an Argon atmosphere was added glacial acetic acid(8.9 μl, 5 eq.) together with tributyltinhydride (24.6 μl, 3 eq) and acatalytic amount of Pd(PPh₃)₄. After 30 minutes the reaction mixturecarefully 1 ml 0.5 M HCl/EtOAc was added to the reaction solution. Theproduct was precipitated by addition of diethyl ether and the whiteprecipitate was collected by means of centrifugation and washed severaltimes with ether. Tert-butanol was added and evaporated again to removean excess of HCl and the resulting product was dissolved in water andfreeze dried yielding 46.9 mg (100%) of the desired prodrug 22.M.P. >192° C. (dec.); ¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 1.15 (s, 3H, 17),1.21 (s, 3H, 16), 1.10–2.00 (m, 9H, CH₂-Lys and CH₃-Ala), 1.67 (s, 3H,19), 1.90 (s, 3H, 18), 2.20 (s, 3H, 10-OAc), 2.43 (s, 3H, 4-OAc), 2.85(m, 4H, benzylic and N—CH₂-Lys), 3.80 (d, 1H, ³J=6.9 Hz, 3), 4.24 (d,1H, ²J=8.4 Hz, 20b), 4.31 (d, 1H, ²J=8.4 Hz, 20a), 4.39 (dd, 1H), 4.56(m, 1H, Hα), 5.68 (m, 1H, Hα), 4.98 (d, 1H, 5), 5.08 (m, 4H, 2benzylic-spacer), 5.43 (d, 1H, ³J=2.7 Hz, 2′), 5.70 (d, 1H, ³J=7.0 Hz,2), 5.97 (m, 1H, 3′), 6.22 (m, 1H, 13), 6.32 (m, 1H, 10), 7.05–7.68 (m,24H, aromatic), 7.71 (d, 1H, ³J=7.2 Hz, aromatic), 8.14 (d, 2H, ³J=7.3Hz, aromatic) ppm; MS (FAB) m/e 1499 (M+H)⁺, 1521 (M+Na)⁺; C₈₁H₉₁N₇O₂₁(.3.7HCl) calculated C, 59.60%; H, 5.85 %; N, 6.01%; measured C, 59.60%;H, 5.88%; N, 5.98%.

Example 17

Synthesis of Fmoc-Trp-Lys(Boc)-OBu 24.

To a solution of 3.00 g (5.73 mmol) Fmoc-Trp-ONSu 23 in drydichloromethane under an argon atmosphere were added at 0° C. 0.791 ml(1.00 equiv) triethylamine and 2.12 g (1.10 equiv) H-Lys (Boc)-OBu.HCl.The mixture was stirred at rt for 5 hours, then dichloromethane wasadded and the organic layer was washed with 10% citric acid, saturatedsodium bicarbonate and water, dried over sodium sulfate and evaporated.The white solid 24 (3.52 g, 86%) was used without further purification.¹H-NMR (300 MHz, CDCl₃): δ 1.10–1.92 (m, 24H, 3 CH₂-Lys and 18 t-Bu),2.80–3.20 (m, 3H, N—CH₂-Lys and CH₂-Trp), 3.52 (d, 1H, CH₂-Trp), 4.19(t, 1H, Fmoc), 4.29–4.82 (m, 5H, 2 Fmoc, 2 Hα and NH), 6.54 (d, H,Aryl), 7.06-7.76 (m, 12H, aromatic) ppm; MS (FAB) m/e 1444 (2M+Na);Anal. (C₄₁H₅₀N₄O₇.4H₂O) C, H, N calculated C, 62.90%; H, 6.30%; N,7.15%; measured C, 63.22%; H, 6.49%; N, 7.13%.

Example 18

Synthesis of Boc-D-Ala-Trp-Lys(oc)-OBu 26.

3.52 g (4.95 mmol) of Fmoc-Trp-Lys(Boc)-OBu 24 was dissolved in 100 mlof dioxane/methanol/2N sodium hydroxide (70/25/5) and stirred at rt for1 hour. The mixture was neutralized with acetic acid (0.570 ml) andorganic solvents were evaporated. Water and dioxane were added and thesolution was freeze dried. Diisopropylether was added and afterfiltration the filtrate was evaporated. The product was dissolved in drydichloromethane and added at 0° C. to a solution of 1.41 g (4.93 mmol)Boc-D-Ala-ONSu 6 and 0.756 ml (1.10 equiv) of triethylamine in drydichloromethane. The mixture was stirred for 16 hours after whichdichloromethane was added. The organic layer was washed with 10% citricacid, saturated sodium bicarbonate and water, and dried over sodiumsulfate and evaporated. The product was purified by means of columnchromatography ((SiO₂—first ethyl acetate/heptane 1/1 and thenCHCl₃/MeOH; 9/1) to afford 2.26 g (3.42 mmol, 69%) of the tripeptide 26as white foam. ¹H-NMR (300 MHz, CDCl₃): δ 0.99–1.90 (m, 36H, 3 CH₂-Lys,CH₃-Ala and 3 t-Bu), 2.80–3.50 (m, 4H, N—CH₂-Lys and 2 CH₂-Trp), 3.99(m, 1H, Hα), 4.33 (m, 1H, Hα), 4.77 (br d, 1H, Hα), 6.90–7.65 (m, 5H,aromatic) ppm; MS (AB) m/e 660 (M+H)⁺, 682 M+Na)⁺; Anal.(C₃₄H₅₃N₅O₈.H₂O)C, H, N calculated C, 60.25%; H, 8.17%; N, 10.33%;measured C, 60.47%; H, 8.08%; N, 9.73%.

Example 19

Synthesis of Aloc-D-Ma-Trp-Lys(Aloc)-OH 28.

2.56 g (4.13 mmol) Boc-D-Ala-Trp-Lys (Boc)-OBu (26) was stirred in asolution of hydrochloric acid in ethyl acetate (3M). After 5 hour thesolvent was evaporated, tert-butanol was added and evaporated twice toremove remaining hydrochloric acid. The product was freeze dried indioxane/water to yield a brown coloured powder.

To a solution of 706 mg (1.61 mmol) D-Ala-Phe-Lys-OH 27 inwater/acetonitrile was added triethylamine until a pH of 9–9.5 wasreached. Then a solution of 1.58 g (2.20 equiv) Aloc-ONSu inacetonitrile was added and the mixture was kept basic by addingtriethylamine. After the pH of the mixture did not alter anymore, a 0.5M solution of hydrochloric acid in ethyl acetate was added until a pH of3 was reached. The mixture was thoroughly extracted withdichloromethane. The organic layer was washed with water, dried (Na₂SO₄)and evaporated. The cream coloured product 28 was used without furtherpurification. ¹H-NMR (300 MH, CDCl₃): δ 1.00–1.80 (m, 9H, 3 CH₂-Lys andCH₂-Ala), 2.80–3.35 (m, 4H, N—CH₂-Lys and CH₂Trp), 4.13 (m, 1H, Hα),4.14 (m, 1H, Hα). 4.30–4.95 (m, 6H, 4 Aloc and 2 Hα), 5.01–5.40 (m, 5H,4 Aloc and Hα), 5.70–6.30 (m, 3H, 2 Aloc and NH), 6.90–7.70 (m, 5H,aromatic) ppm; MS (FAB) m/e 572 (M+H)⁺, 594 (M+Na)⁺; Anal. (C₂₉H₃₇N₅O₈.1½H₂O) calculated C, 56.18%; H, 6.44%; N, 11.70%; measured C, 56.07%; H,6.22%; N, 11.21%.

Example 20

Synthesis of Aloc-D-Ala-Trp-Lys(Aloc)-PABA 29.

A solution of 239 mg (0.419 mmol) Aloc-D-Ala-Trp-Lys(Aloc)-OH 28 wasdissolved in dry tetrahydrofuran under an argon atmosphere and cooled to−40° C. N-methylmorpholine (48.3 μl, 1.05 equiv) andisobutylchloroformate (57.0 μl, 1.05 equiv) were added. The reactionmixture was stirred for 2 hours at a temperature below −30° C. Asolution of 4-aminobenzyl alcohol (51.5 mg, 1.00 equiv) andN-methylmorpholine (50.6 μl, 1.1 equiv) in dry THF was added dropwise tothe reaction mixture. After 2 hours tetrahydrofuran was evaporated anddichloromethane was added. The organic layer was washed with saturatedsodium bicarbonate, a 0.5 N potassium bisulphate solution and brine,dried (Na₂SO₄) and evaporated to afford 265 mg (94%) of the desiredproduct 29 as a cream coloured powder. ¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ1.00–1.62 (m, 9H, CH₃-Ala and 3 CH₂-Lys), 2.90–3.70 (m, 4H, N—CH₂-Lysand CH₂-Trp), 4.48–4.92 (m, 7H, 2 Hα and 4 Aloc), 4.72 (s, 2H, CH₂—OH),5.00–5.50 (m, 5H, 4 Aloc and Hα), 5.35–6.05 (m, 2H, Aloc), 6.80–7.83 (m,9H, aromatic) ppm; MS (FAB) m/e 677 (M+H)⁺, 699 (M+Na)⁺.

Example 21

Synthesis of Aloc-D-Ala-Trp-Lys(Aloc)-PABC-PNP.

To a solution of 384 mg (0.602 mmol) of Aloc-D-Ala-Trp-Lys(Aloc)-PABA 29in dry tetra hydrofuran/dichloromethane under an argon atmosphere,4-nitrophenylchloroformate (182 mg, 1.50 equiv) and dry pyridine (73 μl,1.50 equiv) were added. The mixture was stirred at rt for 48 hours, andthen ethyl acetate was added. The organic layer was washed with 10%citric acid, brine and water, dried Na₂SO₄) and evaporated yielding ayellow solid. The product was purified by means of column chromatography(SiO₂—CHCl₃/MeOH; 30/1) to afford 324 mg (67%) of carbonateAloc-D-Ala-Trp-Lys(Aloc)-PABC-PNP as a cream coloured powder. ¹H-NMR(300 MHz, CDCl₃/CD₃OD): δ 1.00–2.10 (m, 9H, CH₃-Ala and 3 CH₂-Lys),2.90–3.70 (m, 4H, N—CH₂-Lys and CH₂-Trp), 3.64 (m, 1H, Hα), 3.81 (m, 1H,Hα), 4.38–4.81 (m, 5H, Hα and 4 Aloc), 5.10–5.35 (m, 4H, Aloc), 5.21 (s,2H, CH₂—OH), 5.40–6.00 (m, 2H, Aloc), 7.00–7.85 (m, 11H, aromatic), 8.25(d, 2H, J=8.1, aromatic); MS (FAB) m/e 842 (M+H)⁺, 864 (M+Na)⁺.

Example 22

Synthesis of Aloc-D-Ala-Trp-Lys(Aloc)-PABC-PABA 30.

To a solution of 219 mg (260 μmol) of Aloc-D-Ala-Trp-Lys(Aloc)PABC-PNPand 35.2 mg (1.1 equiv) 4-aminobenzyl alcohol in dryN,N-dimethylformamide under an Argon atmosphere was addeddiisopropylethylamine (45.3 μl, 1.00 equiv) and a catalytic amount ofN-hydroxybenzotriazole (10.5 mg, 0.30 equiv). The reaction solution wasstirred for 48 hours after which it was diluted with 10%propanol-2/EtOAc. The organic layer was washed with saturated sodiumbicarbonate, 0.5 N potassium bisulfate and brine, dried over anhydroussodium sulfate and evaporated to dryness. The pale yellow residual filmwas purified by means of column chromatography (SiO₂—CHCl₃/MeOH 15/1) toyield 192 mg (89%) of the desired product 30. ¹H-NMR (300 MHz, CDCl₃): δ0.90–2.10 (m, 9H, CH₃-Ala and 3 CH₂-Lys), 2.90–3.70 (m, 4H, N—CH₂-Lysand CH₂-Trp), 4.08 (m, H, Hα), 4.40–4.86 (m, 6H, 2 benzylic-spacer and 4Aloc), 4.90–5.40 (m, 7H, 2 benzylic-spacer Hα and Aloc), 5.50 (m, 1H,Aloc), 5.92 (m, 1H, Aloc), 6.72–7.82 (m, 13H, aromatic) ppm; MS (FAB)m/e 848 (M+Na)⁺; (C₄₃H₅₁N₇O₁₀ .2¾H₂O) calculated C, 58.99%; H, 6.50%; N,11.20%; measured C, 59.15%; H, 6.25%; N 11.15%.

Example 23

Synthesis of Aloc-D-Ala-Trp-Lys(Aloc)-PABC-PABC-PNP 31.

A solution of 70 mg (85 μmol) of compound 30, pyridine (17 μl, 2.5equiv) and 4-nitrophenylchloroformate (34 mg, 2.0 equiv) was stirredunder an Argon atmosphere at 0° C. for two hours and for 24 hours atroom temperature. The solution was evaporated in vacuo and the residualproduct was dissolved in chloroform. After washing the organic layerwith brine and 0.5 N potassium bisulfate, the organic layer was driedover anhydrous sodium sulfate and concentrated to dryness. The resultingcrude product was subjected to column chromatography (SiO₂—CHCl₃/MeOH20/1) to obtain 54 mg (64%) of 31 as a pale yellow solid. ¹H-NMR (300MHz, CDCl₃/CD₃OD): δ 0.90–2.10 (m, 9H, CH₃-Ala and 3 CH₂-Lys), 2.90–3.10(m, 4H, N—CH₂-Lys and CH₂-Trp), 3.27 (bd, 1H, benzylic), 4.35–4.78 (m,6H, 2Hα and Aloc), 4.90–5.52 (m, 4H, Aloc), 5.13 (s, 2H,benzylic-spacer), 5.60 (m, 1H, Aloc), 5.94 (m, 1H, Aloc), 7.10–7.46 (m,15H, aromatic), 8.36 (d, 2H, aromatic-PNP) ppm; MS (FAB) m/e 991 (M+H)⁺,1013 (M+Na)⁺; C₅₀H₅₄N₅₄N₈O₁₄.¾H₂O) calculated C 59.78%; H, 5.57 %; N,11.15%; measured C, 60.12%; H, 5.89%; N, 10.76%.

Example 24

Synthesis of Aloc-D-Ala-Trp-Lys(Aloc)-PABC-PABC-DOX 32.

The double spacer-containing 4-nitrophenylcarbonate 31 (41 mg, 0.041mmol) and doxorubicin-HCl (26 mg, 1.1 equiv) in N-methylpyrrolidinonewere treated at room temperature with triethylamine (6.3 μl, 1.1 equiv).The reaction mixture was stirred in the dark for 48 hours, againtriethylamine (1.1 equiv) was added and after an additional 24 hours thereaction mixture was diluted with 10% 2-propanol/ethyl acetate. Theorganic layer was washed with water and brine, and was dried (Na₂SO₄).After evaporation of the solvents the crude product was purified bymeans of column chromatography (SiO₂—CHCl₃/MeOH; 9/1) followed bycircular chromatography using a chromatotron supplied with a 2 mm silicaplate (chloroform-methanol; 9/1), to yield 45 mg (78%) of the protectedprodrug 32. ¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 0.92–1.52 (m, 13H, sugarCH₃, CH₃-Ala, 3 CH₂-Lys and 2′), 2.15 (dd, 1H, 8), 2.36 (bd, 1H, 8),3.18 (bd, 1H, 10), 2.90–3.10 (m, 5H, N—CH₂-Lys and CH₂-Trp and 10), 3.59(bs, 1H, 4′), 3.82 (m, 1H, 3′), 3.85 (m, 1H, Hα), 4.11 (s, 3H, OMe),4.21 (m, 1H, Hα), 4.45 (dd, 1H, 5′), 4.30–4.62 (m, 5H, Hα and 4 Aloc),4.76 (s, 2H, 14), 4.96 (s, 2H, benzylic spacer), 5.11 (s, 2H, benzylicspacer), 5.513–5.4 (m, 2H, Aloc), 5.48 (bs, 1H, 1′), 5.58 (m, 2H, Alocand 7), 5.91(m, 2H, Aloc), 6.70–7.39 (m, 11H, aromatic 5 Trp and 6spacers), 7.41 (d, 1H, J=8.4 Hz, 3), 7.63 (d, 2H, aromatic spacer), 7.78(t, 1H, 2), 8.03 (d, 1H, J=7.6 Hz, 1) ppm; MS (FAB) m/e 1417 (M+Na)⁺.

Example 25

Synthesis of D-Ala-Trp-Lys-PABC-PABC-DOX (.7½ HCl) 33.

To a solution of 36 mg (0.026 mmol) protected prodrug 32 in dryTHF/dichloromethane under an argon atmosphere was added morpholine (22μl, 10 equiv) together with a catalytic amount of Pd(PPh₃)₄. Thereaction mixture was stirred for 1 hour in the dark. The red precipitatewas collected by means of centrifugation. Ethyl acetate was added andthe mixture was acidified using 0.5 ml of 1 M hydrochloric acid/ethylacetate. The precipitate was collected by means of centrifugation andwashed several times with ethyl acetate. Tert-butanol was added andevaporated and the resulting red film was freeze dried in water yielding28 mg (72%) of the doxorubicin prodrug 33. ¹H-NMR (300 MHz,CDCl₃/CD₃OD): δ 1.10–1.96 (m, 13H, CH₃-Ala, CH₃-sugar, 3 CH₂-Lys and2′), 2.09 (m, 1H, 8), 2.35 (bd, 1H, J=15.1 Hz, 8), 2.79–3.39 (m, 3H,N—CH₂-Lys, CH₂-Trp and 10), 3.60 (s, 1H, 4′), 4.00 (bs, 1H, 3′), 4.09(s, 3H, OMe), 4.54 (m, 1H, 5′), 4.77 (s, 2H, 14), 4.97 (2*d, 2H, Bnspacer), 5.13 (s, CH₂, Bn spacer), 5.28 (bs, 1H, 1′), 5.48 (bs, 1H, 7),6.99–7.72 (m, 12H, 5 Trp and 6 spacer), 7.62 (d, 1H, 7.6 Hz, 3), 7.55(d, 2H, J=8.2 Hz, aromatic spacer), 7.83 (t, 1H, 2), 8.05 (d, 1H, J=7.7Hz, 1) ppm; MS (FAB) m/e 1228 (M+H)⁺; Anal. (C₆₃H₇₀N₈O₁₈.7 ^(1/2)HCl)calculated C, 50.42%; H, 5.21%; N, 7.47%; measured C, 50.56%; H, 5.48%;N, 7.35%.

Example 26

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-PABA 34.

100 mg (0.105 mmol) of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-PNP 18 wasdissolved in dry N,N-diethylformamide under an argon atmosphere andcooled to −8° C. 4-Aminobenzylalcohol (14.2 mg, 1.1 equiv), dipea (18.3μl, 1.0 equiv) and 1-hydroxybenzotriazole (HOBt) (4 mg, 0.3 equiv) wereadded. The reaction mixture was stirred for 48 hours at roomtemperature, and diluted with 10% 2-propanol/ethyl acetate. The organiclayer was washed with water, saturated sodium bicarbonate, 0.5 Npotassium bisulfate, and brine, dried over sodium sulfate (Na₂SO₄), andevaporated to yield the desired product 34 as a cream colored powder 86mg (88%). ¹H NMR (300 MHz CDCl₃) δ 0.95–2.05 (m, 9H, 3CH₂-Lys andCH₃-Ala), 2.88–3.11 (m, 4H, 2H Bn-Phe and N—CH₂-Lys), 3.95–4.62 (m, 7H,3Hα and 4H Aloc), 4.75 (s, 2H, CH₂—OH), 5.12–5.21 (m, 6H, 4 Aloc andCH₂-Bn), 5.09 (s, 2H, CH₂-Bn), 5.65–6.00 (m, 2H, Aloc), 6.79–7.41 (m,15H, aromatic) 7.62 (d, 2H, aromatic) ppm; MS (FAB) m/e 959 (M+Na)⁺.

Example 27

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-PABC-PNP 35.

To a solution of 59 mg (0.063 mmol) ofAloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-PABA 34 in dry tetrahydrofuran anddichloromethane under an argon atmosphere, were added at −40° C.respectively pyridine (13 μl, 2.5 equiv) and 4-nitrophenyl chloroformate(25 mg, 2.0 equiv). After stirring for 4.5 hours at −40° C. andovernight at 6° C., pyridine (10 μl, 2.0 equiv) and4-nitrophenylchloroformate (25 mg, 2.0 equiv) were added again. This wasrepeated after 48 hours stirring at 6° C. After another 48 hours thesolution was evaporated in vacuo and the residual product was dissolvedin chloroform. The organic layer was washed with 10% citric acid, brineand water, dried over sodium sulfate (Na₂SO₄) and evaporated yielding ayellow solid. The crude product was purified by means of columnchromatography (SiO₂—CHCl₃/MeOH; 15/1) to give the desired product 35quantitatively. ¹H-NMR (300 Mz, CDCl₃/CD₃OD): δ 1.12–1.89 (m, 9H,CH₃-Ala and 3 CH₂-Lys), 3.04 (m, 1H, benzylic), 3.14 (m, 2H, N—CH₂-Lys),3.27 (bd, 1H, benzylic), 4.09 (m, 1H, Hα), 4.28 (m, 1H, Hα), 4.34–4.68(m, 5H, Hα and Aloc), 5.02–5.40 (m, 4H, Aloc), 5.14 (s, 2H,benzylic-spacer), 5.21 (s, 2H, benzylic-spacer), 5.31 (s, 2H, benzylicspacer), 5.72 (m, 1H, Aloc), 5.90 (m, 1H, Aloc), 7.10–7.52 (m, 17H,aromatic), 7.63 (d, 2H, J=8.3 Hz, aromatic), 8.27 (d, 2H, J=9.1 Hz,aromatic-PNP) ppm; MS (FAB) me 1102 (M+H)⁺, 1124 (M+Na)⁺.

Example 28

Synthesis of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-PABC-DOX 36.

The 4-nitrophenyl carbonate 35 (69 mg, 0.063 mmol) and doxorubicin-HCl(40 mg, 1.1 equiv) in N-methylpyrrolidinone were treated at roomtemperature with triethylamine (9.7 μl, 1.1 equiv). The reaction mixturewas stirred in the dark for 24 hours and the reaction mixture wasdiluted with 10% 2-propanol/ethyl acetate. The organic layer was washedwith water and brine, and was dried over sodium sulfate (Na₂SO₄). Afterevaporation of the solvents the crude product was purified by means ofcolumn chromatography (SiO₂—CHCl₃/MeOH 9/1) followed by circularchromatography using a chromatotron supplied with a 2 mm silica plate(CHCl₃/MeOH; 9/1), to yield 65 mg (71%) of the protected prodrug 36.¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 1.10–1.80 (m, 14H, sugar CH₃, CH₃-Ala,3 CH₂-Lys and 2′), 2.14 (dd, 1H, 8), 2.36 (bd, 1H, 8), 3.18 (bd, 1H,10), 2.82–3.41 (m, 6H, benzylic Phe and N—CH₂-Lys and 10), 3.37 (s, 1H,4′), 3.60 (bs, 1H, 3′), 4.02 (m, 1H, Hα), α), 4.07 (s, 3H, OMe) 4.29(dd, 1H, 5′), 4.37–4.68 (m, 5H, Hα and 4 Aloc), 4.76 (s, 2H, 14), 4.95(bs, 2H, benzylic spacer), 5.10 (s, 2H, benzylic spacer), 5.14 (s, 2H,benzylic spacer), 5.02–5.35 (m, 4H, Aloc), 5.27 (bs, 1H, 1′), 5.47 (bs,1H, 7), 5.70 (m, 1H, Aloc), 5.89 (m, 1H, Aloc), 7.09–7.50 (m, 16H, 5 Pheand 10 spacers and 3), 7.64 (d, 2H, J=8.4 Hz, 2H aromatic spacer), 7.79(t, 1H, J=8.1 Hz, 2), 8.06 (d, 1H, J=7.5 Hz, 1) ppm; MS (FAB) m/e 1506(M+H)⁺, 1528 (M+Na)⁺.

Example 29

Synthesis of D-Ala-Phe-Lys-PABC-PABC-PABC-DOX (.2HCl) 37.

To a solution of 40 mg protected prodrug 36 (0.027 mmol) in drytetrahydrofuran/dichloromethane under an argon atmosphere were addedmorpholine (24 μl, 10 equiv) and a catalytic amount of Pd(PPh₃)₄. Thereaction mixture was stirred for 1 hour in the dark. The red precipitatewas collected by means of centrifugation and washed several times withethyl acetate. Water and dioxane were added and the mixture wasacidified using 4.4 ml of 0.125 mM hydrochloric acid. After freezedrying 26 mg (70%) of doxorubicin prodrug 37 was obtained. ¹H-NMR (300MHz, CDCl₃/CD₃OD): δ 1.19 (d, 3H, J=6.9 Hz, sugar CH₃), 1.27 (d, 3H,J=6.6 Hz, CH₃-Ala), 1.25–2.00 (m, 8H, 3 CH₂-Lys and 2′), 2.18 (dd, 1H,8), 2.33 (br d, 1H, J=16.1 Hz, 8), 2.89–3.38 (m, 6H, N—CH₂-Lys and 10and Bn Phe), 3.60 (s, 1H, 4′), 3.72 (m, 1H, 3′), 4.08 (s, 3H, OMe), 4.18(m, 1H, Hα), 4.53 (dd, 1H, 5′) 4.66 (m, 1H, Hα), 4.77 (s, 2H, 14), 4.96(s, 2H, Bn spacer), 5.11 (s, 2H, bn spacer), 5.17 (s, 2H, Bn spacer),5.27 (br s, 1H 1′), 5.48 (br s, 1H, 7), 7.05–7.35 (m, 16H, aromaticspacer and 3), 7.52 (d, 2H, J=8.5 Hz, aromatic spacer), 7.84 (t, 1H, 2),8.01 (d, 1H, J=7.7 Hz, 1) ppm.

Example 30

Synthesis of 2′-[4-nitrophenyl carbonate]-paclitaxel 38.

To a solution of 194 mg (0.227 mmol) paclitaxel in dry dichloromethaneunder an Argon atmosphere was added pyridine (4 drops). At −50° C., 275mg (6.0 eq.) 4-nitrophenyl chloroformate dissolved in drydichloromethane was added. The reaction mixture was stirred at −50° C.and after 4 hours 4-nitrophenyl chloroformate (4.2 eq.) was added. After1 hour the mixture was diluted with dichloromethane and washed with 0.5N potassium bisulfate and brine and dried over anhydrous sodiumsulphate. After evaporation of the solvents the residual yellow film waspurified by means of column chromatography (SiO₂—EtOAc/Hex 1/1), toyield 133 mg of activated paclitaxel 38 (78%, 73% conversion). M.P. 161°C.; ¹H-NMR (300 MHz, CDCl₃): δ 1.14 (s, 3H, 17), 1.25 (s, 3H, 16), 1.68(s, 3H, 19), 1.92 (s, 3H, 18), 2.22 (s, 3H, 10-OAc), 2.49 (s, 3H,4-OAc), 2.55 (m, 1H, 6a), 3.82 (d, 1H, 3), 4.21 (d, 1H, 20b), 4.32 (d,1H, 20a), 4.42 (m, 1H, 7), 4.96 (bd, 1H, 5), 5.53 (d, 1H, 2′), 5.69 (d,1H, 2), 6.09 (q, 1H, 3′), 6.29 (s, 1H, 10), 6.34 (m, 1H, 13), 6.90 (d,1H, N—H), 7.20–7.65 (m, 13H, aromatic), 7.75 (d, 2H, aromatic), 8.15 (d,2H, aromatic), 8.25 (d, 2H, nitrophenyl) ppm; MS (FAB) m/e 1020 (M+H)⁺,1042 (M+Na)⁺; C₅₄H₅₄N₂O₁₉ (.1½H₂O) calculated C, 62.00%; N, 2.68%;measured C, 61.89%; H, 5.52%; N, 2.64%.

Example 31

Synthesis of 2′-[H-D-Ala-Phe Lys-PABC-N(Me)-(CH₂)₂—N(Me)CO]-paclitaxel(.2HCl) 43.

Step a: Synthesis of N(Me)—(CH₂)₂—N(Me)—Z 39 (Z=benzyloxycarbonyl).

To a solution of 1.21 g (13.7 mmol) N,N′-dimethyl ethylenediamine in drydichloromethane under an Argon atmosphere at room temperature was addeddropwise a solution of Z-ONSu (338 mg, 1.36 mmol) in drydichloromethane. After stirring for 120 minutes the solution wasconcentrated in vacuo. The residual product was dissolved in ethylacetate and the organic layer was washed with brine. The organic solventwas dried over anhydrous sodium sulfate and evaporated to dryness. Theoily product was purified by means of column chromatography(SiO₂—CHCl₃/MeOH 1/1) to obtain 249 mg (83%) of the product 39 as anoil. ¹H-NMR (300 MHz, CDCl₃): δ 2.42 (bd, 3H, ³J=13.9 Hz, CH ₃—NH—CH₂),2.73 (m, 2H, CH₃—NH—CH ₂), 2.95 (s, 3H, CH₃—N), 3.41 (bs, 2H, CH₂—N),5.13 (s, 2H, CH₂—Z), 7.25–7.40 (m, 5H, aromatic) ppm.

Step b: Synthesis of 2′-[Z—N(Me)—(CH₂)₂—N(Me)CO]-paclitaxel 40.

To a solution of 114 mg (112 μmol) 2′-activated paclitaxel 38 and 25 mgZ-protected N,N′-dimethyl ethylenediamine 39 in dry dichloromethaneunder an Argon atmosphere at −50° C. was added triethyl amine (20.0 μl,144 μmol). The solution was stirred 7 hours at −40° C., subsequentlyallowed to heat up to room temperature and then stirring was continuedovernight at room temperature. The solution was diluted withdichloromethane and washed with saturated sodium bicarbonate, brine and0.5 N potassium bisulfate. The organic layer was dried over anhydroussodium sulfate and concentrated in vacuo to obtain a yellow film. Theproduct was purified by column chromatography (SiO₂—EtOAc/Hex 2/1) toobtain 113 mg (92%) of the desired product 40. M.P. 130–131° C.; ¹H-NMR(300 MHz, CDCl₃): δ 1.12 (s, 3H, 17), 1.21 (s, 3H, 16), 1.70 (s, 3H,19), 2.00 (s, 3H, 18), 2.26 (s, 3H, 10-OAc), 2.60 (s, 3H, 4-OAc), 2.90(s, 3H, CH₃-spacer), 2.94 (s, 3H, CH₃-spacer), 2.97 (m, 1H, CH₂-spacer),3.06 (m, 1H, CH₂-spacer), 3.54 (m, 1H, CH₂-spacer), 3.78 (m, 1H,CH₂-spacer), 3.84 (d, 1H, ³J=7.2 Hz, 3), 4.23 (d, 1H, ²J=8.4 Hz, 20b),4.32 (d, 1H, ²J=8.4 Hz, 20a), 4.47 (m, 1H, 7), 4.69 (d, 1H, ²J=12.4 Hz,benzylic), 4.85 (d, 1H, ²J=12.4 Hz, benzylic), 5.01 (m, 1H, 5), 5.47 (d,1H, ³J=2.9 Hz, 2′), 5.68 (d, 1H, ₃J=7.0 Hz, 2), 6.19 (dd, 1H, ³J=9.8 Hz,³J=2.9 Hz, 3′), 6.28 (s, 1H, 10), 6.33 (m, 1H, 13), 6.94–7.70 (m, 16H,aromatic), 7.83 (d, 2H, ³J=7.3 Hz, aromatic), 8.16 (d, 2H, ³J=7.1 Hz,aromatic), 8.57 (d, 1H, ³J=9.8 Hz, NH) ppm; MS (FAB) m/e 1102 (M+H)⁺,1124 (M+Na)⁺; C₆₀H₆₇N₃O₁₇ (.H₂O) calculated C, 64.33%; H, 6.21%; N,3.73%; measured C, 64.65%; H, 6.11%; N, 3.76%.

Step c: Synthesis of 2′-[N(Me)—(CH₂)₂—N(Me)CO]-paclitaxel (.7AcOH) 41.

To a solution of 61.8 mg (56.1 μmol) of 40 in 5% acetic acid/methanolwas added a catalytic amount of 10% Pd—C. The mixture was stirred for 1hour under a H₂ atmosphere. The Pd—C was removed by means ofcentrifugation, methanol was evaporated in vacuo, and ethyl acetate wasadded. The organic layer was extracted with water. The water layer wasfreeze dried yielding 78.0 mg (100%) of the desired product 41.

Step d: Synthesis of2′-[Aloc-D-Ala-Phe-Lys(Aloc)-PABC-N(Me)—(CH₂)₂—N(Me)CO]-paclitaxel 42.

To a solution of 152 mg (95.8 μmol) of paclitaxel-spacer compound 41 and80.7 mg (101 μmol) of carbonate 16 in dry tetrahydrofuran under an Argonatmosphere was added triethyl amine (200 μl, 1.44 mmol). After 24 hoursthe solution was concentrated to dryness and the residual product wasdissolved in dichloromethane and washed with saturated sodiumbicarbonate and brine. The organic layer was dried over anhydrous sodiumsulfate and evaporated in vacuo. The crude product was subjected tocolumn chromatography (SiO₂—EtOAc/Hex/MeOH 5/5/1) to obtain 113 mg (72%)of the desired protected prodrug 42. M.P. 127–128° C.; ¹H-NMR (300 Mz,CDCl₃): δ 1.13 (s, 3H, 17), 1.22 (s, 3H, 16), 1.27 (d, 3H, ³J=5.6 Hz,CH₃-Ala), 1.04–2.00 (m, 6H, CH₂-Lys), 1.69 (s, 3H, 19), 2.00 (s, 3H,18), 2.22 (s, 3H, 10-OAc), 2.59 (s, 3H, 4-OAc), 2.90 (s, 3H,CH₃-spacer), 2.91 (s, 3H, CH₃-spacer), 2.76–3.46 (m, 6H, CH₂-spacer,benzylic and N—CH₂-Lys), 3.54. (m, 1H, CH₂-spacer), 3.74 (m, 1H,CH₂-spacer), 3.84 (d, 1H, ³J=7.0 Hz, 3), 4.00–5.00 (m, 3H, 3 Hα), 4.23(d, 1H, ²J=8.4 H 20b), 4.32 (d, 1H, ²J=8.4 Hz, 20a), 4.48 (m, 1H, 7),4.62 (d, 1H, ²J=12.3 Hz, benzylic), 4.83 (d, 1H, ²J=12.4 Hz, benzylic),4.30–4.73 (m, 4H, Aloc), 4.93–5.39 (m, 5H, Aloc and 5), 5.48 (d, 1H,³J=2.9 Hz, 2′), 5.69 (d, 1H, ³J=7.0 Hz, 2), 5.54–5.78 (m, 1H, Aloc),5.88 (m, 1H, Aloc), 6.18 (bd, 1H, 3′), 6.30 (s, 1H, 10), 6.33 (m, 1H,13), 7.05–7.78 (m, 20H, aromatic), 7.82 (d, 2H, ³J=7.4 Hz, aromatic),8.16 (d, 2H, ³J=7.2 Hz, aromatic) ppm; MS (FAB) m/e 1653 (M+Na)⁺;C₈₆H₁₀₂N₈O₂₄ calculated C, 62.61%; H, 6.35%; N 6.79%; measured C,62.40%; H 6.31%; N, 6.36%.

Step e: Synthesis of2′-[H-D-Ala-Phe-Lys-PABC-N(Me)—(CH₂)₂—N(Me)CO]-paclitaxel (.2HCl) 43.

To a solution of 83.0 mg (50.9 μmol) protected prodrug 42 in drytetrahydrofuran under an Argon atmosphere was added glacial acetic acid(12 μl, 4.0 eq.) together with tributyltinhydride (41 μl, 3.0 eq) and acatalytic amount of Pd(PPh₃)₄. After 30 minutes the product wasprecipitated by addition of diethyl ether. The white precipitate wascollected by means of centrifugation and washed several times withdiethyl ether. Tert-butanol was added and evaporated again to remove anexcess of HCl and the resulting product was dissolved in water/dioxaneand freeze dried yielding 56 mg (70%) of prodrug 43. M.P. 142° C.;¹H-NMR (300 MHz, CDCl₃): δ 1.13 (s, 3H, 17), 1.21 (s, 3H, 16), 1.26 (d,3H, ³J=6.6 Hz, CH₃—Ala), 1.05–2.00 (m, 6H CH₂-Lys), 1.69 (s, 3H, 19),2.00 (s, 3H, 18), 2.22 (s, 3H, 10-OAc), 2.58 (s, 3H, 4-OAc), 2.89 (s,3H, CH₃-spacer), 2.91 (s, 3H, CH₃-spacer), 2.67–3.64 (m, 3H,Ch₂-spacer), 2.95 (m, 1H, benzylic), 3.07 (m, 2H, N—CH₂-Lys), 3.15 (m,1H, benzylic), 3.78 (m, 1H, CH₂-spacer), 3.83 (d, 1H, ³J=7.1 Hz, 3),4.10–5.05(m, 2H, 2 Hα), 4.22 (d, 1H, ²J=8.4 Hz, 20b), 4.32 (d, 1H,²J=8.4 Hz, 20a), 4.46 (m, 1H, 7), 4.60 (m, 1H, Hα), 4.65 (d, 1H, ²J=12.3Hz, benzylic-spacer), 4.80 (d, 1H, ²J=12.4 Hz, benzylic-spacer), 4.99(bd, 5H, ³J=7.4 Hz, 5), 5.47 (d, 1H, ³J=2.9 Hz, 2′), 5.68 (d, 1H, ³J=6.9Hz, 2), 6.17 (bd, 1H, ³J=2.9 Hz, ³J=9.6 Hz, 3′), 6.30 (s, 1H, 10), 6.31(m, 1H, 13), 7.05–7.70 (m, 20H, aromatic), 7.82 (d, 2H, ³J=7.5 Hz,aromatic), 8.16 (d, 2H, ³J=7.2 Hz, aromatic), 8.54 (d, 1H, ³J=9.6 Hz,NH-paclitaxel) ppm; MS (FAB) m/e 1463 (M+H)⁺, 1485 (M+Na)⁺; C₈₅H₉₇N₇O₂₂(.3AcOH) calculated C 61.04%; H, 6.50%; N, 6.71%; measured C, 60.91%; H,6.45%; N, 7.10%.

Example 32

Synthesis of2′-O-[D-Ala-Phe-Lys-PABC-PABC-N(Me):CH—N(Me)CO]paclitaxel.2HCl.

Step a: Synthesis of2′-O-[Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-N(Me)—(CH₂)₂—N(Me)CO]paclitaxel.

To a solution of paclitaxel-spacer conjugate 41 (50 mg, 48.6 μmol) andpeptide-spacer conjugate 18 (46.3 mg, 48.6 μmol) in THF (3 mL) was addedtriethylamine (101 μL, 0.730 mmol). The reaction mixture was stirred atroom temperature for 15 h and then concentrated under reduced pressure.The residue was dissolved in dichloromethane, and the solution waswashed with a saturated aqueous NaHCO₃ solution and brine, dried overNa₂SO₄, filtered, and concentrated under reduced pressure. Columnchromatography (SiO₂—EtOAc/Hex/MeOH 5/4/1) gave 44 (58.2 mg, 32.7 μmol,67%).

¹H-NMR (300 MHz, CDCl₃): δ 1.12 (s, 3H, 17), 1.21 (s, 3H, 16), 1.26 (d,3H, J=6.6 Hz, CH₃ of Ala), 1.05–2.00 (m, 6H, 3×CH₂ of Lys), 1.69 (s, 3H,19), 1.99 (s, 3H, 18), 2.22 (s, 3H, 10-OAc), 2.58 (s, 3H, 4-OAc), 2.90(s, 3H, N—CH₃), 2.91 (s, 3H, N—CH₃), 2.80–3.85 (m, 9H, N—CH₂—CH₂—N andCH₂ of Phe and N—CH₂ of Lys and 3), 4.00–5.38 (m, 19H, 3×Hα and 20 and 7and 2×CH₂ of spacer and 5 and 2×CH ₂═CH—CH ₂), 5.46 (d, 1H, J=2.7 Hz,2′), 5.60 (m, 1H, CH₂═CH—CH₂), 5.69 (d, 1H, J=6.9 Hz, 2), 5.89 (m, 1H,CH₂═CH—CH₂), 6.16 (dd, 1H, J=9.3 Hz, J=2.4 Hz, 3′), 6.30 (s, 1H, 10),6.31 (m, 1H, 13), 7.09–7.81 (m, 26H, aromatic), 8.16 (d, 2H, J=7.2 Hz,aromatic) ppm.

Step b: Synthesis of2′-O-[D-Ala-Phe-Lys-PABC-PABC-N(Me)-(CH₂)₂—N(Me)CO]paclitaxel.2HCl.

To a solution of protected prodrug 44 (50.0 mg, 28.1 μmol) in dry THF (3mL) were added tributyltin hydride (22.7 μL), Pd(PPh₃)₄ (6.5 mg, 5.6μmol), and acetic acid (6.5 μL, 0.112 mmol). After 30 min, the reactionmixture was slowly added to cold diethyl ether. The white precipitatewas collected by means of centrifugation and washed two times withdiethyl ether. The residue was suspended in ethyl acetate and a 0.5 MHCl solution in ethyl acetate (0.5 mL) was added under vigorousstirring. The white precipitate was collected by means of centrifugationand washed two times with ethyl acetate. tert-Butyl alcohol was added tothe residue and subsequently evaporated to remove excess HCl. Theresidue was dissolved in water en freeze-dried, giving 45 (32.0 mg, 19.0μmol, 68%).

¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 1.17 (s, 3H, 17), 1.19(s, 3H, 16), 1.26(d, 3H, J=6.9 Hz, CH₃ of Ala), 1.20–2.00 (m, 6H, 3×CH₂ of Lys), 1.70 (s,3H, 19), 2.02 (s, 3H, 18 ), 2.21 (s, 3H, 10-OAc), 2.55 (s, 3H, 4-OAc),2.80–2.99 (m, 4H, CH₂ of Phe and N—CH₂ of Lys), 2.93 (s, 6H, 2×N—CH₃),3.10–3.87 (m, 4H, N—CH₂—CH₂—N), 3.86 (d, 1H, J=6.9 Hz, 3), 4.06 (q, 1H,J=7.0 Hz, Hα), 4.26–4.76 & 5.02 (m 8H, 2×Hα and 20 and 7 and CH₂ ofspacer and 5), 5.16 (s, 2H, CH₂ of spacer), 5.45 (d, 1H, J=2.5 Hz, 2′),5.72 (d, 1H, J=7.1 Hz, 2), 6.10 (m, 1H, 3′), 6.26 (m, 1H, 13), 6.40 (s,1H, 10), 7.08–7.62 (m, 24H, aromatic), 7.77 (d, 2H, J=7.6 Hz, aromatic),8.14 (m, 2H, aromatic) ppm.

Example 33

Synthesis of2′-O-[D-Ala-Phe-Lys-PACC-N(Me)—(CH₂)₂—N(Me)CO]paclitaxel-2HCl 49.

Step a: Preparation of NZ-D-Ala-Phe-Lys(NZ)—OH 9c.

To a solution of tripeptide 8 (506 mg, 1.39 mmol) in dichloromethane (10mL) were added trimethylsilyl chloride (0.568 mL, 4.44 mmol) and DIPEA(0.509 mL, 2.92 mmol). The reaction mixture was stirred at refluxtemperature for 1.5 h. Then, the reaction mixture was cooled down to 0°C., after which DIPEA (776 μL, 4.44 mmol) and para-nitrobenzylchloroformate (NZ—Cl) (629 mg, 2.92 mmol) were added. The reactionmixture was stirred at room temperature for 5 h and then concentratedunder reduced pressure. The residue was partitioned between ethylacetate and acetate buffer (pH=5). The organic layer was washed withmore acetate buffer, water, and brine, dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. Column chromatography(SiO₂—CH₂Cl₂/MeOH/AcOH 90/7/5) gave 9c (620 mg, 0.858 mmol, 61%).

¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 1.18 (d, 3H, J=7.1 Hz, CH₃ of Ala),1.36–1.97 (m, 6H,3×CH₂ of Lys), 2.94 (dd, 1H, CH₂ of Phe), 3.12–3.23 (m,3H, CH₂ of Phe and N—CH₂ of Lys), 4.12 (m, 1H, Hα), 4.42 (m, 1H, Hα),4.63 (m, 1H, Hα), 5.17 (m, 4H, CH₂ of NZ), 7.17–7.25 (m, 5H, aromatic),7.51 (d, 4H, J=8.2 Hz, aromatic), 8.19 (d, 4H, J=8.0 Hz, aromatic) ppm.

Step b: Preparation of NZ-D-Ala-Phe-Lys(Z)-PACA 46.

To a solution of protected tripeptide 9c (300 mg, 0.415 mmol) in THF (10mL) were added at −40° C. N-methylmorpholine (50.2 μL, 0.457 mmol) andisobutyl chloroformate (62.4 mg, 0.457 mmol). The reaction mixture wasstirred at −30° C. for 3 h. Then, spacer 10 (77.3 mg, 0.498 mmol) andN-methylmorpholine (55.0 μL, 0.498 mmol) in THF (5 nL) were added. Thereaction mixture was stirred for 15 h, the reaction temperature slowlybeing raised to room temperature. The reaction mixture was concentratedunder reduced pressure and the residue was dissolved in dichloromethane.The solution was washed with a saturated aqueous NaHCO₃ solution, a 0.5M aqueous KHSO₄ solution, and brine, dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. Column chromatography(SiO₂—CHCl₃/MeOH 93/7) gave 46 (267 mg, 0.313 mmol, 75%).

¹H-NMR (300 MHz, CDCl₃/CD₃OD): δ 1.22 (d, 3H, J=7.2 Hz, CH₃ of Ala),1.40–2.04 (m, 6H, 3×CH₂ of Lys), 2.96 (dd, 1H, J=9.9 Hz, J=14.4 Hz, CH₂of Phe), 3.15 (m, 2H, N—CH₂ of Lys), 3.30 (dd, 1H, J=14.4 Hz, J=4.2 Hz,CH₂ of Phe), 4.08 (m, 1H, Hα), 4.23 (dd, 2H, J=1.1 Hz, J=5.6 Hz, CH₂ ofspacer), 4.46 (m, 1H, Hα), 4.56 (m, 1H, Hα), 4.90 (d, 1H, J=13.8 Hz, CH₂of NZ), 4.99 (d, 1H, J=13.8 Hz, CH₂ of NZ), 5.17 (s, 2H, CH₂ of NZ),6.26 (dt, 1H, J=5.7 Hz, J=15.9 Hz, CH═CH—CH₂), 6.52 (d, 1H, J=15.9 Hz,CH═CH—CH₂), 7.22–7.32 (m, 9H, aromatic), 7.53 (d, 2H, J=8.6 Hz,aromatic), 7.61 (d, 2H, J=8.6 Hz, aromatic), 8.04 (d, 2H, J=8.6 Hz,aromatic), 8.19 (d, 2H, J=8.6 Hz, aromatic) ppm.

Step c: Preparation of NZ-D-Ala-Phe-Lys(NZ)-PACC-PNP 47.

To a solution of peptide-spacer 46 (244 mg, 0.286 mmol) in THF (15 mL)were added DIPEA (0.216 mL, 1.24 mmol), para-nitrophenyl chloroformate(187 mg, 0.927 mmol), and pyridine (6.3 μL, 77.2 μmol). The reactionmixture was stirred for 48 h and then diluted with ethyl acetate (50mL). The solution was washed with a 10% aqueous citric acid solution,water, and brine, dried over Na₂SO₄, filtered, and concentrated underreduced pressure. Column chromatography (SiO₂—CHCl₃/MeOH 95/5) gave 47(291 mg, 0.286 mmol, 100% A).

¹H-NMR (300 MHz, CDCl₃/CD₃OD/DMSO-₆): δ 1.23 (d, 3H, J=7.1 Hz, CH₃ ofAla), 1.38–2.03 (m, 6H, 3×CH₂ of Lys), 2.97 (dd, 1H, J=9.9 Hz, J=14.0Hz, CH₂ of Phe), 3.14 (m, 2H, N—CH₂ of Lys), 3.28 (dd, 1H, CH₂ of Phe),4.10 (m, 1H, Hα), 4.47 (m, 1H, Hα), 4.54 (m, 1H, Hα), 4.87–5.00 (m, 4H,CH₂ of spacer and CH₂ of NZ), 5.17 (s, 2H, CH₂ of NZ), 6.27 (dt, 1H,J=6.8 Hz, J=15.4 Hz, CH═CH—CH₂), 6.70 (d, 1H, J=15.4 Hz, CH═CH—CH₂),7.22–7.33 (m, 9H, aromatic), 7.47 (d, 2H, J=9.2 Hz, aromatic), 7.53 (d,2H, J=8.6 Hz, aromatic), 7.67 (d, 2H, J=8.6 Hz, aromatic), 8.04 (d, 2H,J=8.6 Hz, aromatic), 8.18 (d, 2H, J=8.6 Hz, aromatic), 8.30 (d, 2H,J=9.2 Hz, aromatic) ppm.

Step d: Preparation of2′-O-[NZ-D-Ala-Phe-Lys(NZ)-PACC-N(Me)—(CH₂)₂—N(Me)CO]paclitaxel 48.

To a solution of paclitaxel-spacer conjugate 41 (50 mg, 48.6 μmol) andpeptide-spacer conjugate 47 (54.5 mg, 53.5 μmol) in THF (3 mL) was addedtriethylamine (101 μL, 0.730 mmol). The reaction mixture was stirred atroom temperature for 15 h and then concentrated under reduced pressured.The residue was dissolved in dichloromethane, and the solution waswashed with a saturated aqueous NaHCO₃ solution and brine, dried overNa₂SO₄, filtered, and concentrated under reduced pressure. Columnchromatography (SiO₂—EtOAc/Hex/MeOH 5/4/1) gave 48 (65.7 mg, 35.6 μmol,73%).

¹H-NMR (300 MHz, CDCl₃): δ 1.12 (s, 3H, 17), 1.21 (s, 3H, 16), 1.30 (d,3H, J=7.0 Hz, CH₃ of Ala), 1.20–2.10 (m, 6H, 3×CH₂ of Lys), 1.68 (s, 3H,19), 2.00 (s, 3H, 18), 2.22 (s, 3H, 10-OAc), 2.58 (s, 3H, 4-OAc), 2.91(s, 3H, N—CH₃), 2.95 (s, 3H, N—CH₃), 2.90–3.90 (m, 8H, N—CH₂—CH₂—N andCH₂ of Phe and N—CH₂ of Lys), 3.83 (d, 1H, J=6.6 Hz, 3), 4.10–5.00 (m,11H, 3×Hα and 20 and 5 and 7 and CH═CH—CH ₂ and CH₂ of NZ), 5.16 (s, 2H,CH₂ of NZ), 5.46 (d, 1H, J=3.3 Hz, 2′), 5.68 (d, 1H, J=7.5 Hz, 2),6.01–6.17 (m, 2H, 3′ and CH═CH—CH₂), 6.31 (s, 1H, 10), 6.32 (m, 1H, 13),6.40 (d, 1H, J=15.9 Hz, CH═CH—CH₂), 7.19–7.70 (m, 24H, aromatic), 7.83(d, 2H, J=7.2 Hz, aromatic), 8.01 (d, 2H, J=8.7 Hz, aromatic), 8.15–8.19(m, 4H, aromatic) ppm.

Step e: Preparation of2′-O-[D-Ala-Phe-Lys-PACC-N(Me)—(CH₂)₂—N(Me)CO]paclitaxel.2HOAc 49.

To a solution of protected prodrug 48 (50.0 mg, 27.1 μmol) in methanol(5 mL) was added acetic acid (1.5 mL) and zinc (88.5 mg, 1.35 mmol). Theresultant suspension was stirred at room temperature for 24 h. Then, thereaction mixture was filtered over HYFLO. Water was added and methanolwas evaporated under reduced pressure. The resultant solution wasfreeze-dried to obtain a slightly yellow solid, which was dissolved in amixture of dichloromethane and methanol (2 mL, 1:1 v/v). This solutionwas added to cold diisopropyl ether. The white precipitate was collectedby means of centrifugation and washed two times with diisopropyl ether,which afforded prodrug 49 (27.8 mg, 17.3 μmol, 64%).

Example 34

Stability of Both Double Spacer Containing Paclitaxel Prodrugs 22 and43.

The prodrugs were incubated at concentrations of 150 μM in 0.1 MTris/HCl buffer (pH 7.3) for 3 days and showed no formation ofdegradation products (TTC, RP₁₈; CH₃CN/H₂O/AcOH 19/19/2).

Stability of the Double Spacer Containing Doxorubicin Prodrug 20.

The prodrug was incubated at a concentration of 100–270 μM in 0.1 MTris/HCl buffer (pH 7.3) for 90 hours and showed no formation ofdegradation products (TLC, RP₁₈; CH₃CN/H₂O/AcOH 19/19/2).

Example 35

Enzymatic Hydrolysis of the Double Spacer Containing Prodrugs byPlasmin.

Hydrolysis of the doxorubicin prodrugs was investigated by incubation ata prodrug concentration of 100 μM in 0.1 M Tris/hydrochloric acid buffer(pH 7.3) in the presence of 50 or 20 μg/mL human plasmin (Fluka).Analysis was carried out with the following HPLC system using aChrompack Microsphere-C18 column (3 μm, 2×100×4.6 1 mm). Elution of theanalytical column was performed using 7:3 methanol/50 mM Et₃N-formatebuffer (pH 3.0). Detection was performed using an UV-detector (λ=500nm).

[prodrug] (μM) [plasmin] (μg/mL) T_(1/2) activation (min) Prodrug 50 10050 19 Prodrug 50 200 20 >75 Prodrug 20 200 20 12

Hydrolysis of the paclitaxel prodrugs was investigated by incubation ata prodrug concentration of 200 μM in 0.1 M Tris/hydrochloric acid buffer(pH 7.3) in the presence of 100 μg/mL human plasmin (Fluka). All doublespacer containing paclitaxel prodrugs were converted to yield thecorresponding parent drug. Capillary electrophoresis was carried outwith a CE Ext. Light Path Capillary (80.5 cm, 50 μm), with 1:1methanol/0.05 M sodium phosphate buffer (pH 7.0) as eluent Detection wasperformed at 200 and 254 nm.

[prodrug] [plasmin] T_(1/2) activation T_(1/2) cyclisation (μM) (μg/mL)(min) (min) Prodrug 51 200 100 42 Prodrug 43 200 100 4 47 Prodrug 22 200100 7.5

Example 36

Cytotoxicity.

The anti-proliferative effect of prodrugs and parent drugs wasdetermined in vitro applying seven well-characterised human tumor celllines and the microculture sulphorhodamine B (SRB) test. Theanti-proliferative effects were determined and expressed as IC₅₀ values,that are the (pro)drug concentrations that gave 50% inhibition whencompared to control cell growth after 5 days of incubation.

TABLE 1 ID₅₀ values^(a,b) (ng/ml) of prodrugs and parent drugs. EVSA-Cell Line: MCF-7 T WIDR IGROV M19 A498 H226 Prodrug 20 242 546 627 896302 2303 503 Prodrug 43 60 119 117 499 96 681 62 Prodrug 22 11 5 5 22 725 7 Paclitaxel <3 <3 <3 10 <3 <3 <3 Doxorubicin 10 8 11 60 16 90 199^(a)Drug dose that inhibited cell growth by 50% compared to untreatedcontrol cultures. ^(b)SRB cell viability test. Cell lines: MCF-7; breastcancer. EVSA-T; breast cancer. WIDR; colon cancer. IGROV; ovariancancer. M19; melanoma. A498; renal cancer. H226; non-small cell lungcancer.

1. A compound of the formula:V—(W)_(k)—(X)_(l)—A—Z wherein: V is an enzymatically removablespecifier, optionally being removable after prior binding to a receptor;(W)_(k)—(X)_(l)—A is an elongated self-eliminating spacer system; W andX are each a 1,(4+2n) electronic cascade self-eliminating spacer, beingthe same or different; A is either a spacer group of formula (Y)_(m),wherein Y is a 1,(4+2n) electronic cascade self-eliminating spacer, or agroup of formula U being a cyclisation self-elimination spacer; Z is atherapeutic or diagnostic moiety; k and l are independently an integerfrom 0 (included) to 5 (included); m is an integer from 1 (included) to5 (included); n is an integer from 0 (included) to 10 (included), andk+1>0.
 2. The compound of claim 1, wherein group U is an ω-aminoaminocarbonyl cyclisation spacer, Z is a moiety bearing a hydroxylgroup, and Z is bonded to U via the hydroxyl group of Z.
 3. The compoundof claim 1, wherein the electronic cascade spacers W, X and Y areindependently selected from compounds having the formula:

whereinQ=—R⁵C═CR⁶—, S, O, NR⁵, —R⁵C═N—, or —N═CR⁵—P=NR⁷, O, S a, b, and c are independently an integer of 0 to 5; I, F andG are independently selected from compounds having the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ independently representH, C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy(OH), amino (NH₂), mono-substituted amino (NR_(x)H), di-substitutedamino (NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me,CONHMe, cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl,morpholino, thiol (SH), thioether (SR_(x)), tetrazole, carboxy (COOH),carboxylate (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)),sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)),sulphinyl (S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate(OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² are independentlyselected from a C₁₋₆ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀aryl group, two or more of the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, or R⁹ optionally being connected to one another to form one or morealiphatic or aromatic cyclic structures.
 4. The compound of claim 2,wherein A is an ω-amino aminocarbonyl cyclisation elimination spacer Uand U is a moiety having the formula:

wherein: a is an integer of 0 or 1; and b is an integer of 0 or 1; and cis an integer of 0 or 1; provided that a+b+c=2 or 3; and wherein R¹and/or R² independently represent H, C₁₋₆ alkyl, said alkyl beingoptionally substituted with one or more of the following groups: hydroxy(OH), ether (OR_(x)), amino (NH₂), mono-substituted amino (NR_(x)H),di-substituted amino (NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN,CONH₂, SO₂Me, CONHMe, cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆alkylpiperazinyl, morpholino, thiol (SH), thioether (SR_(x)), tetrazole,carboxy (COOH), carboxylate (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate(S(═O)₂OR_(x)), sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate(S(═O)OR_(x)), sulphinyl (S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), andphosphate (OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² areselected from a C₁₋₆ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀aryl group; and R³, R⁴, R⁵, R⁶, R⁷, and R⁸ independently represent H,C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy (OH),amino (NH₂), mono-substituted amino (NR_(x)H), di-substituted amino(NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe,cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino,thiol (SH), thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate(COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)), sulphonyl(S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)), sulphinyl(S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate(OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² are selected from aC₁₋₆ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀ aryl group; andwherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ can be a part of one or morealiphatic or aromatic cyclic structures, two or more of the substituentsR¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ optionally being connected to oneanother to form one or more aliphatic or aromatic cyclic structures. 5.The compound of claim 1 wherein A is an electronic cascade spacer havingthe structural formula:

whereinQ=—R⁵C═CR⁶—, S, O, NR⁵, —R⁵C═N—, or —N═CR⁵—P=NR⁷, O, S a, b, and c are independently an integer of 0 to 5; I, F andG are independently selected from compounds having the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ independently representH, C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy(OH), amino (NH₂), mono-substituted amino (NR_(x)H), di-substitutedamino (NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me,CONHMe, cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl,morpholino, thiol (SH), thioether (SR_(x)), tetrazole, carboxy (COOH),carboxylate (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR),sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)),sulphinyl (S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate(OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² are independentlyselected from a C₁₋₄ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀aryl group, two or more of the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, or R⁹ optionally being connected to one another to form one or morealiphatic or aromatic cyclic structures.
 6. The compound of claim 5wherein Z is a moiety bearing a phenolic hydroxyl group, and Z is bondedto A via the phenolic hydroxyl group of Z.
 7. The compound of claim 1wherein the specifier V contains a substrate that can be cleaved byplasmin.
 8. The compound of claim 1 wherein the specifier V contains asubstrate that can be cleaved by a cathepsin.
 9. The compound of claim 1wherein the specifier V contains a substrate that can be cleaved bycathepsin B.
 10. The compound of claim 1 wherein the specifier Vcontains a substrate that can be cleaved by β-glucuronidase,prostate-specific antigen (PSA), urokinase-type plasminogen activator(u-PA), or a member of the family of matrix metalloproteinases.
 11. Thecompound of claim 1 to 5 wherein the specifier V contains anitro-(hetero)aromatic moiety that can be removed by reduction underhypoxic conditions or by reduction by a nitroreductase.
 12. The compoundof claim 1 wherein the spacer system (W)_(k)—(X)_(l)—A isp-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminobenzyloxycarbonyl-p-amino-benzyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminobenzyloxycarbonyl-p-aminocinnamyloxycarbonyl,p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl,p-aminophenylpentadienyloxycarbonyl-p-aminocinnamyloxycarbonylp-aminophenylpentadienyloxycarbonyl-p-aminobenzyloxycarbonyl,p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyloxycarbonyl,p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminobenzyloxycarbonyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycaxbonyl(methylamino)ethyl(methylamino)carbonyl,p-aminobenzyloxycarbonyl-p-aminobenzyl,p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyl,p-aminocinnamyloxycarbonyl-p-aminobenzyl, p-aminobenzyloxycarbonylp-aminocinnamyl, p-aminocinnamyloxycarbonyl-p-aminocinnamyl,p-aminophenylpentadienyloxycarbonyl-p-aminocinnamyl,p-aminophenylpentadienyloxycarbonyl-p-aminobenzyl, orp-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyl.
 13. Thecompound of claim 1 wherein the moiety Z is an anticancer agent.
 14. Thecompound of claim 13 wherein the said moiety Z is the amino containingcytotoxic moiety daunorubicin, doxorubicin,N-(5,5-diacetoxypentyl)doxorubicin, an anthracycline, mitomycin C,mitomycin A, 9-amino camptothecin, aminopterin, actinomycin, bleomycin,N⁸-acetyl spermidine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine,tallysomycin, or derivatives thereof, the hydroxyl containing cytotoxicmoiety etoposide, camptothecin, irinotecan, topotecan, 9-aminocamptothecin, paclitaxel, docetaxel, esperamycin,1,8-dihydroxy-bicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one, anguidine,doxorubicin, morpholine-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, vincristine, vinblastine, or derivatives thereof, thesulfhydryl containing cytotoxic moiety esperamicin, 6-mercaptopurine, orderivatives thereof, the carboxyl containing cytotoxic moietymethotrexate, camptothecin (ring-opened form of the lactone), butyricacid, retinoic acid, or derivatives thereof.
 15. The compound of claim 1wherein the moiety Z represents the anticancer drug paclitaxel or apaclitaxel derivative that is coupled to the promoietyV—(W)_(k)—(X)_(l)—U— via its 2′-hydroxyl group.
 16. The compound ofclaim 1 wherein the specifier V is a tripeptide.
 17. The compound ofclaim 16 wherein the covalent linkage of said tripeptide specifiermoiety to its immediately adjacent moiety is at the C-terminus of saidtripeptide specifier moiety.
 18. The compound of claim 17 wherein theC-terminal amino acid residue is arginine or lysine, the middle aminoacid residue is selected from the group consisting of alanine, valine,leucine, isoleucine, methionine, phenylalanine, cyclohexylglycine,tryptophan and proline, and the N-terminal amino acid residue is aD-amino acid residue, a protected L-amino acid residue, or protectedglycine.
 19. The compound of claim 1 wherein the specifier V isD-alanylphenylalanyllysine, D-valylleucyllysine, D-alanylleucyllysine,D-valylphenylalanyllysine, D-valyltryptophanyllysine, orD-alanyltryptophanyllysine.
 20. The compound of claim 1 wherein thespecifier V is an amino-terminal capped peptide covalently linked at itsC-terminus to its immediately adjacent moiety.
 21. The compound of claim20 wherein the said amino-terminal capped peptide specifier isbenzyloxycarbonylphenylalanyllysine, benzyloxycarbonylvalyllysine,D-phenylalanylphenylalanyllysine, benzyloxycarbonylvalyllysine,tertbutyloxycarbonylphenylalanyllysine, benzyloxycarbonylalanylarginine,benzyloxycarbonylphenylalanyl-N-tosylarginine,2-aminoethylthiosuccinimidopropionylvalinylcitrulline,2-aminoethylthiosuccinimidopropionyllysylphenylalanyllysine,acetylphenylalanyllysine, orbenzyloxycarbonylphenylalanyl-O-benzoylthreonine.
 22. The compound ofclaim 1 which isN-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)doxorubicin,N-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-doxorubicin,N-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxy-carbonyl)daunorubicin,N-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-amino-benzyloxycarbonyl-p-aminobenzyloxycarbonyl)daunorubicin,N-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)mitomycinC,N-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-mitomycinC,N-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,N-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)camptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)camptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)etoposide,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)etoposide,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)irinotecan,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)irinotecan,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)topotecan,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)topotecan,N-(D-alanylphenylalanyllysyl-p-aminocinnamyloxy-carbonyl-p-aminobenzyloxycarbonyl)doxorubicin,N-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)daunorubicin,N-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)mitomycinC,N-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,2′-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,7-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,2′-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,7-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,O-(D-alanylphenylalanyllysyl-p-aminocinnamyl-oxycarbonyl-p-aminobenzyloxycarbonyl)camptothecin,O-(D-alanylphenylalanyllysyl-p-amino-cinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)etoposide,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)irinotecan,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)topotecan,N-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)doxorubicin,N-(D-alanylphenylalanyllysyl-p-amino-cinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)daunorubicin,N-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)mitomycinC,N-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)-9-aminocamptothecin,2′-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)paclitaxel,7-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)paclitaxel,2′-O-(D-alanylphenylalanyllysyl-p-amino-cinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)docetaxel,7-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)docetaxel,O-(D-alanylphenyl-alanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)camptothecin,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)-9-aminocamptothecin,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)etoposide,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)irinotecan,O-(D-alanylphenylalanyllysyl-p-aminocinnamyl-oxycarbonyl-p-aminocinnamyloxycarbonyl)topotecan,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-docetaxel,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)camptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-9-aminocamptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)etoposide,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)irinotecan,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)topotecan,2′-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,7-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,2′-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,7-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)camptothecin,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-9-aminocamptothecin,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)etoposide,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)irinotecan,O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)topotecan,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,7-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)camptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-9-aminocamptothecin,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)etoposide,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)irinotecan,O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)topotecan,N-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxy-carbonyl)doxorubicin,N-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxy-carbonyl-p-aminobenzyloxycarbonyl)doxorubicin,N-(D-valylleucyllysyl-p-aminobenzyloxy-carbonyl-p-aminobenzyloxycarbonyl)daunorubicin,N-(D-valylleucyllysyl-p-aminobenzyloxy-carbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)daunorubicin,N-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)mitomycinC,N-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)mitomycinC,N-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyl-oxycarbonyl)-9-aminocamptothecin,N-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,2′-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,2′-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxy-carbonyl)paclitaxel,7-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxy-carbonyl)paclitaxel,7-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxy-carbonyl-p-aminobenzyloxycarbonyl)paclitaxel,2′-O-(D-valylleucyllysyl-p-aminobenzyloxy-carbonyl-p-aminobenzyloxycarbonyl)docetaxel,2′-O-(D-valylleucyllysyl-p-aminobenzyloxy-carbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,7-O-(D-valyl-leucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,7-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)camptothecin,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)camptothecin,O-(D-valylleucyllysyl-p-aminobenzyl-oxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-amino-camptothecin,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)-etoposide,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)etoposide,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)irinotecan,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)irinotecan,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)topotecan,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)topotecan,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)doxorubicin,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)daunorubicin,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)mitomycinC,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,2′-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,7-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)paclitaxel,2′-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,7-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)docetaxel,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)camptothecin,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)-9-aminocamptothecin,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)etoposide,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)irinotecan,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl)topotecan,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)doxorubicin,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)daunorubicin,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)mitomycinC,N-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)-9-amino-camptothecin,2′-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)paclitaxel,7-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)paclitaxel,2′-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)docetaxel,7-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)docetaxel,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)camptothecin,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)-9-aminocamptothecin,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)etoposide,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)irinotecan,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl)topotecan,2′-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,7-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,2′-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,7-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)camptothecin,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-9-aminocamptothecin,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)etoposide,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)irinotecan,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)topotecan,2′-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,7-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-paclitaxel,2′-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,7-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl-(methylamino)ethyl(methylamino)carbonyl)docetaxel,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)camptothecin,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-9-aminocamptothecin,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)etoposide,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)irinotecan,O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)topotecan,2′-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,7-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel,2′-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,7-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)docetaxel,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)camptothecin,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)-9-aminocamptothecin,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)etoposide,O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)irinotecan,O-(D-valyl-leucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)topotecan,or a salt thereof.
 23. The compound of claim 22 which isN-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)doxorubicin.24. The compound of claim 22 which isN-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl)doxorubicin.25. The compound of claim 22 which is2′-O-(D-alanylphenylalanyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel.26. The compound of claim 22 which is2′-O-(D-valylleucyllysyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel.27. The compound of claim 22 which is2′-O-(D-alanylphenylalanyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel.28. The compound of claim 22 which is2′-O-(D-valylleucyllysyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl)paclitaxel.29. The compound of claim 1 wherein the moiety Z is an antibiotic, ananti-inflammatory agent, or an anti-viral agent.
 30. The compound ofclaim 1 wherein the specifier V contains a polymer.
 31. The compound ofclaim 1, wherein the specifier V is removed by an enzyme that istransported to the vicinity of target cells or target tissue viaantibody-directed enzyme prodrug therapy (“ADEPT”), polymer-directedenzyme prodrug therapy (“PDEPT”), virus-directed enzyme prodrug therapy(“VDEPT”) or gene-directed enzyme prodrug therapy (“GDEPT”).
 32. Aprocess for preparing a pharmaceutical composition in a solid or aliquid formulation for administration orally, topically or by injection,said process comprising mixing a compound according to claim 1 with apharmaceutically acceptable carrier.
 33. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 34. The compound of claim 1 wherein the specifier V contains amoiety capable of targeting the compound to the target site byselectively complexing with a receptor or other receptive moietyassociated with a target cell population.
 35. The compound of claim 1wherein the specifier V contains an antigen-recognizing immunoglobulinor an antigen-recognizing fragment of an immunoglobulin.